Koichi Mitsuya

Continuous EGFR-TKI administration following radiotherapy for non-small cell lung cancer patients with isolated CNS failure.

INTRODUCTION Based on previous reports, patients who experience isolated central nervous system (CNS) failure may not have systemic acquired resistance to EGFR-TKI therapy. However, because there are few articles that have reported on the clinical efficacy of continuous EGFR-TKI administration following progressive disease (PD) in isolated CNS metastasis, we retrospectively investigated the possibility of using the treatment. PATIENTS AND METHODS From July 2002 to December 2009, 17 non-small cell lung cancer patients showed isolated CNS failure after clinical benefit (partial response or stable disease longer than 6 months) from EGFR-TKIs and continuously received EGFR-TKIs following radiotherapy (whole brain radiotherapy or stereotactic radiotherapy) to the CNS metastases. RESULTS The response rate and the disease control rate of CNS lesions were 41% and 76%, respectively. The median progression free survival, extracranial progression free survival and the median overall survival time were 80 days, 171 days and 403 days, respectively. The toxicities which were observed during the first EGFR-TKI treatments were sustained, but did not worsen during this study period. The acute toxicities caused by radiotherapy to the CNS were controllable. There were no remarkable late toxicities related to the treatment. CONCLUSIONS Continuous administration of EGFR-TKI following radiotherapy after PD in isolated CNS metastasis appears to be a valid treatment option.

α-type-1 polarized dendritic cell-based vaccination in recurrent high-grade glioma: a phase I clinical trial.

BackgroundHigh-grade gliomas including glioblastoma multiforme (GBM) are among the most malignant and aggressive of tumors, and have a very poor prognosis despite a temozolomide-based intensive treatment. Therefore, a novel therapeutic approach to controlling recurrence is needed. In the present study, we investigated the effect of activated dendritic cell (DC) (α-type-1 polarized DC)-based immunotherapy on high-grade glioma patients with the HLA-A2 or A24 genotype.MethodsNine patients with recurrent high-grade gliomas including 7 with GBMs who fulfilled eligibility criteria were enrolled into a phase I study of monocyte-derived DC-based immunotherapy. HLA-genotyping revealed 1 case of HLA-A*0201 and 8 cases of A*2402. Enriched monocytes obtained using OptiPrepTM from leukapheresis products on day1, were incubated with GM-CSF and IL-4 in a closed serum-free system, and activated on day6 with TNF-α, IL-1β, IFN-α, IFN-γ, and poly I/C. After pulsing with a cocktail of 5 synthetic peptides (WT-1, HER2, MAGE-A3, and MAGE-A1 or gp100) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Thawed DCs were injected intradermally in the posterior neck at a dose per cohort of 1.0, 2.0 and 5.0× 107/body.ResultsThe frequency of CD14+ monocytes increased to 44.6% from 11.9% after gradient centrifugation. After a 7-day-incubation with cytokines, the mean percentage of DCs rated as lin-HLA-DR+ in patients was 56.2 ± 19.1%. Most DCs expressed high levels of maturation markers, co-stimulatory molecules and type-1 phenotype (CD11c+HLA-DR+) with a DC1/2 ratio of 35.6. The amount of IL-12 produced from activated DCs was 1025 ± 443 pg/ml per 105 cells. All 76 DC injections were well tolerated except for transient liver dysfunction with grade II. Six patients showed positive immunological responses to peptides in an ELISPOT assay, and positive skin tests to peptide-pulsed DC and KLH were recognized in 4 cases. The clinical response to DC injections was as follows :1 SD and 8 PD. Interestingly, the SD patient, given 24 DC injections, showed a long-term recurrence-free and immunological positive response period.ConclusionsThese results indicate peptide cocktail-treated activated α-type-1 DC-based immunotherapy to be a potential therapeutic tool against recurrent high-grade glioma with mainly HLA-A*2402.Trial registrationCurrent non-randomized investigational trial UMIN-CTR UMIN ID: 000000914.

Effect of the STAT3 inhibitor STX-0119 on the proliferation of cancer stem-like cells derived from recurrent glioblastoma.

Signal transducer and activator of transcription (STAT) 3, a member of a family of DNA-binding molecules, is a potential target in the treatment of cancer. The highly phosphorylated STAT3 in cancer cells contributes to numerous physiological and oncogenic signaling pathways. Furthermore, a significant association between STAT3 signaling and glioblastoma multiforme stem-like cell (GBM-SC) development and maintenance has been demonstrated in recent studies. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the present study, we focused on cancer stem-like cells derived from recurrent GBM patients and investigated the efficacy of STX-0119. Three GBM stem cell lines showed many stem cell markers such as CD133, EGFR, Nanog, Olig2, nestin and Yamanaka factors (c-myc, KLF4, Oct3/4 and SOX2) compared with parental cell lines. These cell lines also formed tumors in vivo and had similar histological to surgically resected tumors. STAT3 phosphorylation was activated more in the GBM-SC lines than serum-derived GB cell lines. The growth inhibitory effect of STX-0119 on GBM-SCs was moderate (IC50 15-44 µM) and stronger compared to that of WP1066 in two cell lines. On the other hand, the effect of temozolomide was weak in all the cell lines (IC50 53-226 µM). Notably, STX-0119 demonstrated strong inhibition of the expression of STAT3 target genes (c-myc, survivin, cyclin D1, HIF-1α and VEGF) and stem cell-associated genes (CD44, Nanog, nestin and CD133) as well as the induction of apoptosis in one stem-like cell line. Interestingly, VEGFR2 mRNA was also remarkably inhibited by STX-0119. In a model using transplantable stem-like cell lines in vivo GB-SCC010 and 026, STX-0119 inhibited the growth of GBM-SCs at 80 mg/kg. STX-0119, an inhibitor of STAT3, may serve as a novel therapeutic compound against GBM-SCs even in temozolomide-resistant GBM patients and has the potential for GBM-SC-specific therapeutics in combination with temozolomide plus radiation therapy.

Feasibility of proton beam therapy for chordoma and chondrosarcoma of the skull base.

We explored the general feasibility of proton beam therapy for chordoma and chondrosarcoma of the skull base. Clinical records and treatment-planning data of patients with the pathological diagnosis of chordoma or chondrosarcoma were examined. Proton beam therapy was administered for gross tumor mass as well as microscopic residual disease after surgery. The prescribed dose was determined to maximize the coverage of the target and to not exceed predefined constraints for the organs at risk. Eight cases of chordoma and eight cases of chondrosarcoma were enrolled. The median tumor volume was 40 cm(3) (range, 7 to 546 cm(3)). The prescribed dose ranged from 50 to 70 Gy (relative biological effectiveness [RBE]), with a median of 63 Gy RBE. The median follow-up duration was 42 months (range 9 to 80 months). The overall survival rate was 100%, and the local control rate at 3 years of chordoma and chondrosarcoma were 100% and 86%. None of the patients developed radiation-induced optic neuropathy, brain stem injury, or other severe toxicity. Proton beam therapy is generally feasible for both chordoma and chondrosarcoma of the skull base, with excellent local control and survival rates.

YKL-40 downregulation is a key factor to overcome temozolomide resistance in a glioblastoma cell line

The frequent recurrence of glioblastoma multiforme (GBM) after standard treatment with temozolomide (TMZ) is a crucial issue to be solved in the clinical field. O6‑methylguanine‑DNA methyltransferase (MGMT) is considered one of the major mechanisms involved in TMZ resistance. However, some important mechanisms for TMZ resistance other than MGMT have recently been identified. In the present study, we established a TMZ-resistant (TMZ-R) U87 glioblastoma cell line in vitro and in vivo and investigated novel targeting molecules other than MGMT in those cells. The TMZ-R U87 glioblastoma cell line was established in vitro and in vivo. TMZ-R U87 cells showed a more invasive activity and a shorter survival time in vivo. Gene expression analysis using DNA microarray and quantitative PCR (qPCR) demonstrated that YKL‑40, MAGEC1 and MGMT mRNA expression was upregulated 100-, 83- and 6-fold, respectively in the TMZ-R U87 cell line. Western blot analysis and qPCR demonstrated that STAT3 phosphorylation, STAT3 target genes and stem cell and mesenchymal marker genes were upregulated to a greater extent in the TMZ‑resistant cell line. Notably, short hairpin (sh)RNA‑based inhibition against the YKL‑40 gene resulted in moderate growth inhibition in the resistant cells in vitro and in vivo. Additionally, YKL‑40 gene inhibition exhibited significant suppression of the invasive activity and particularly partially restored the sensitivity to TMZ. Therefore, YKL‑40 may be a novel key molecule in addition to MGMT, that is responsible for TMZ resistance in glioblastoma cell lines and could be a new target to overcome TMZ resistance in recurrent glioblastomas in the future.

Effect of the STAT3 inhibitor STX-0119 on the proliferation of a temozolomide-resistant glioblastoma cell line

Glioblastoma multiforme (GBM) is one of the most malignant and aggressive tumors and has a very poor prognosis, with a median survival time of less than 2 years. Once recurrence develops, there are few therapeutic approaches to control the growth of glioblastoma. In particular, temozolomide (TMZ)-resistant (TMZ-R) GBM is very difficult to treat, and a novel approach to overcome resistance is eagerly awaited. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the current study, the efficacy of STX-0119 was evaluated against our established TMZ-resistant U87 cell line using quantitative PCR-based gene expression analysis, in vitro assay and animal experiments. The growth inhibitory effect of STX-0119 on U87 and TMZ-R U87 cells was moderate (IC₅₀, 34 and 45 µM, respectively). In particular, STX-0119 did not show significant inhibition of U87 tumor growth; however, it suppressed the growth of the TMZ-R U87 tumor in nude mice by more than 50%, and prolonged the median survival time compared to the control group. Quantitative PCR revealed that YKL-40, MAGEC1, MGMT, several EMT genes, mesenchymal genes and STAT3 target genes were upregulated, but most of those genes were downregulated by STX-0119 treatment. Furthermore, the invasive activity of TMZ-R U87 cells was significantly inhibited by STX-0119. YKL-40 levels in TMZ-R U87 cells and their supernatants were significantly decreased by STX-0119 administration. These results suggest that STX-0119 is an efficient therapeutic to overcome TMZ resistance in recurrent GBM tumors, and could be the next promising compound leading to survival prolongation, and YKL-40 may be a possible surrogate marker for STAT3 targeting.

Novel cancer-testis antigen expression on glioma cell lines derived from high-grade glioma patients

Glioblastoma multiforme (GBM) is one of the most malignant and aggressive tumors, and has a very poor prognosis with a mean survival time of <2 years, despite intensive treatment using chemo-radiation. Therefore, novel therapeutic approaches including immunotherapy have been developed against GBM. For the purpose of identifying novel target antigens contributing to GBM treatment, we developed 17 primary glioma cell lines derived from high-grade glioma patients, and analyzed the expression of various tumor antigens and glioma-associated markers using a quantitative PCR and immunohistochemistry (IHC). A quantitative PCR using 54 cancer-testis (CT) antigen-specific primers showed that 36 CT antigens were positive in at least 1 of 17 serum-derived cell lines, and 17 antigens were positive in >50% cell lines. Impressively, 6 genes (BAGE, MAGE-A12, CASC5, CTAGE1, DDX43 and IL-13RA2) were detected in all cell lines. The expression of other 13 glioma-associated antigens than CT genes were also investigated, and 10 genes were detected in >70% cell lines. The expression of CT antigen and glioma-associated antigen genes with a high frequency were also verified in IHC analysis. Moreover, a relationship of antigen gene expressions with a high frequency to overall survival was investigated using the Repository of Molecular Brain Neoplasia Data (REMBRANDT) database of the National Cancer Institute, and expression of 6 genes including IL-13RA2 was inversely correlated to overall survival time. Furthermore, 4 genes including DDX43, TDRD1, HER2 and gp100 were identified as MGMT-relevant factors. In the present study, several CT antigen including novel genes were detected in high-grade glioma primary cell lines, which might contribute to developing novel immunotherapy and glioma-specific biomarkers in future.

Antitumor Effect of Programmed Death-1 (PD-1) Blockade in Humanized the NOG-MHC Double Knockout Mouse.

Purpose: Humanized mouse models using NOD/Shi-scid-IL2rγnull (NOG) and NOD/LtSz-scid IL2rγnull (NSG) mouse are associated with several limitations, such as long incubation time for stem cell engraftment and the development of xenograft versus host disease in mice injected with peripheral blood mononuclear cells (PBMCs). To solve problems, we used humanized major histocompatibility class I- and class II-deficient NOG mice (referred to as NOG-dKO) to evaluate the antitumor effect of anti-programmed death-1 (PD-1) antibody. Experimental Design: Humanized NOG-dKO mice, in which human PBMCs and human lymphoma cell line SCC-3, or glioblastoma cell line U87 were transplanted, were used as an immunotherapy model to investigate the effect of anti-PD-1 antibody. A biosimilar anti-PD-1 mAb generated in our laboratory was administered to humanized NOG-dKO mice transplanted with tumors. Results: Within 4 weeks after transplantation, human CD45+ cells in antibody-treated mice constituted approximately 70% of spleen cells. The injection of anti-PD-1 antibody reduced by more 50% the size of SCC-3 and U87 tumors. In addition, induction of CTLs against SCC-3 cells and upregulation of natural killer cell activity was observed in the antibody-treated group. Tumor-infiltrating lymphocyte profiling showed that more exhausted marker (PD1+TIM3+LAG3+) positive T cells maintained in anti-PD-1 antibody–treated tumor. A greater number of CD8+ and granzyme-producing T cells infiltrated the tumor in mice treated with the anti-PD-1 antibody. Conclusions: These results suggest that NOG-dKO mice might serve as a good humanized immunotherapy model to evaluate the efficacy of anti-PD-1 antibody prior to the clinical treatment. Clin Cancer Res; 23(1); 149–58. ©2016 AACR.

Elevated preoperative neutrophil-to-lymphocyte ratio as a predictor of worse survival after resection in patients with brain metastasis

OBJECTIVE The median postoperative survival duration of patients with brain metastases who undergo tumor resection is 12 months. Most of these patients die of systemic metastases or the progression of primary cancer but not brain metastases. The criteria for indicating resection are still controversial. Systemic assessment is restricted, especially in patients who need emergent management for their large-size or life-threatening brain metastases. The neutrophil-to-lymphocyte ratio (NLR) is reported to correlate with survival time or progression-free survival in patients with various cancers. However, NLR has not been assessed in patients with brain metastases. The impact of NLR on the survival data of patients who had undergone resection of brain metastases was retrospectively analyzed. METHODS The clinical records of patients who underwent resection of brain metastases at Shizuoka Cancer Center between May 2007 and January 2015 were retrospectively analyzed. NLR was calculated by using the data obtained from the latest examination before resection and before the administration of steroid therapy. Kaplan-Meier curves were used for the assessment of overall survival (OS). RESULTS Of the 105 patients included, 67 patients were male. The median age of the patients was 63 years (range 36-90 years). The primary cancers were lung, colon, breast, uterus, and other cancers in 48, 19, 10, 8, and 20 patients, respectively. The postresection median survival duration was 12 months (range 1-91 months) for the entire series. The optimum NLR threshold value was identified as 5. A significant difference in the median OS was found: 14 months for 82 patients with an NLR < 5 versus 5 months for 23 patients with an NLR ≥ 5 (p = 0.001). CONCLUSIONS In this study, an elevated preoperative NLR is a predictor of worse survival after resection for brain metastases. NLR is a simple, systemic marker and can be used in clinical settings for decision making regarding resection in patients with brain metastases.

A Novel Graft Material for Preventing Cerebrospinal Fluid Leakage in Skull Base Reconstruction: Technical Note of Perifascial Areolar Tissue

Objectives Perifascial areolar tissue (PAT), a layer of loose connective tissue on the deep fascias with a rich vascular plexus, serves as a vital cover over defects with scarce vascularity. We report the usefulness of PAT as a nonvascularized alternative to flaps for reconstruction of dural defects in skull base surgery and transsphenoidal surgery while evaluating its effect on control of cerebrospinal fluid (CSF) leakage. Design A retrospective chart analysis was performed on patients who had undergone repair of a dural defect with PAT during skull base surgery or transsphenoidal surgery between December 2004 and October 2011. Results Twenty-one patients were included: 11 patients had received surgical treatment and/or irradiation. Fourteen of the 21 patients had pre- and/or intraoperative CSF leakage. Only one patient (4.8%) had postoperative CSF leakage requiring additional surgical repair. Ten patients underwent postoperative irradiation from 1 to 15 months after transplant of the PAT. None of the patients had postoperative CSF leakage after irradiation. Conclusion We successfully repaired dural defects using PAT in skull base surgery and transsphenoidal surgery, even in patients with a history of multiple operations and radiotherapy. PAT may serve as a valuable tool for skull base reconstruction.