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Resistance to immune-checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold-, myeloid cell-, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell-subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC.

Supplementary figures, tables, material and methods, and references

Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. In this study, we identified G-protein–coupled receptor family C group 5 member D (GPRC5D) expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, not at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. In addition, we constructed IgG-based anti-GPRC5D/CD3 bispecific T-cell–redirecting antibodies (GPRC5D TRAB), which suppressed the tumor growth of GPRC5D-positive myeloma cells through the activation of T cells in vitro and in vivo in xenograft models. Collectively, these findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy.

Supplementary Tables S1-S2. Supplementary Table S1: Patients' characteristics for flow cytometry analyses on malignant cells from multiple myeloma. Supplementary Table S2: Sequence information of GPA0018 TRAB and GPA0039 TRAB.

Supplementary Figures S1-S7. Figure S1: Cell surface expression of GPRC5D on malignant cells from multiple myeloma (patient number: MM#2 to MM#11 in Supplementary Table S1). Figure S2: Cell surface expression of GPRC5D on cancer cell lines and cytotoxicity of GPRC5D TRABs when co-culturing GPRC5D-expressing NCI-H929 cells without PBMC. Figure S3: Cytotoxicity of GPRC5D TRABs when co-culturing GPRC5D-expressing NCI-H929 cells with CD4+ T cells or CD8+ T cells. Figure S4: Antitumor activity and plasma concentrations of GPA0039 TRAB in mouse models with NCI-H929 tumors. Figure S5: T-cell activation of GPRC5D TRABs after co-culturing with GloResponse{trade mark, serif} NFAT-luc2 Jurkat cells and NCI-H929 cells or Human Bone Marrow CD34+ cells (StemCell Technologies) from two different donors. Figure S6: mRNA expression (TPM value) of GPRC5D and BCMA using the GTEx sample set. Figure S7: Binding activity of 100 nM of anti-GPRC5D antibodies on CHO cells expressing cynomolgus monkey GPRC5D.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy.Significance:Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1

New cancer characteristics can be discovered by focusing on the process of tumor formation. Cancer stem cells (CSCs) are a key subpopulation, as they are theorized to be at the apex of the tumor hierarchy. We can better understand their function in the tumor hierarchy by using sectioned samples to observe the growth of tumors from their origins as CSCs. In this study, we evaluated the growth of moderate differentiated colorectal cancer from LGR5-positive cells, which is a CSC marker of colorectal cancer, using xenograft and three-dimensional culture models spatiotemporally. These cells express LGR5 at high levels and show CSC phenotypes. To detect them, we used a previously generated antibody that specifically targets LGR5, and were therefore able to observe LGR5-positive cells aggregating into small clusters (sCLs) over the course of tumor growth. Because these LGR5-expressing sCLs formed continuously during growth mainly in the invasive front, we concluded that the structure must contribute significantly to the expansion of CSCs and to tumor growth overall. We confirmed the formation of sCLs from gland structures using a three-dimensional culture model. In addition, sCLs exhibited upregulated genes related to stress response and partial/hybrid epithelial-mesenchymal transition (EMT), as well as genes reported to be prognosis factors. Finally, sCLs with high LGR5 expression were identified in clinical samples. Based on these results, we elucidate how sCLs are an important contributors to tumor growth and the expansion of CSCs.

Recurrence after chemotherapy is one of the biggest obstacles in cancer therapy, along with metastasis. Although histopathological evaluation in preclinical models like the xenograft model help us to understand the pathophysiological process of tumor growth, there are not enough detailed histopathological analyses of such models. In this study, to learn how a tumor recovers the typical tumor structure after structural corruption during chemo-treatment, xenografted tumors originating from a patient-derived xenograft of colorectal cancer (CRC) were analyzed histopathologically over time. There were many Duct (Flattened) at Day 1 (one day after the final administration of Irinotecan), but the ratio of Duct (Columnar) and Cribriform-structures typically found in colorectal adenocarcinoma-increased over time. Finally, at Day 15 (15 days after the final administration of Irinotecan), tumor structure and size were once again the same as in the control group. LGR5, a known cancer stem cell (CSC) marker for CRC, was highly expressed on protruding structures observed from Duct (Flattened) during their transformation into Duct (Columnar) and Cribriform. In addition, these LGR5-expressing protruding structures were either Ki67 negative or positive. These results suggest that the formation of protruding structures on Duct (Flattened) is a pivotal first step in the regrowth of tumors.

Background: Blockade of interlukine-6 (IL-6) and programmed death-1-ligand 1 (PD-L1) limits tumour progression in murine models of pancreatic cancer. When PD-L1 expression of metastatic pancreatic cancer (MPC) is increased under the influence of gemcitabine exposure plus IL-6 inhibition, the spectrum of benefit from immune checkpoint blockade may expand to MPC patients with combined blockade of IL-6 and PD-L1 plus gemcitabine-based chemotherapy. We sought to determine PD-L1 expression of liver metastasis during cycle 1 of a phase I study, tocilizumab (TCZ) plus gemcitabine/nab-paclitaxel (GN) in patient with GN-refractory MPC. Methods: This phase 1 study enrolled 10 patients with MPC that had progressed after GN. All patients experienced cycle 1 of TCZ plus GN, TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. Biopsy of liver metastasis was performed before Cycle 1 and at the end of Cycle 1. Tumour microenvironment including PD-L1 expression were evaluated in paired-biopsied specimen from 7 patients by multicolour immunofluorescence staining. Tumour cell and PD-L1 expression were labelled by anti-cytokeratin 19 antibody and anti-PD-L1 antibody clone SP263, respectively. Cell numbers per millimetre squared (cell density) and the percentage of PD-L1 positive cell in tumor area were calculated according to cell types including tumour cell as cytokeratin 19 positive cell and macrophage as CD68 positive cell. Results: Cell density and the percentage of PD-L1 positive tumor cell were 29/mm2 and 1.1% in mean at baseline and tended to be increased up to 142/mm2 (P = 0.11) and 5.2% (P = 0.06) at the end of cycle 1. Means of PD-L1 positive macrophages were 28/mm2 and 6.9% at baseline and were increased to 69/mm2 (P = 0.01) and 14% (P = 0.08). When region of interest was selected from tumor stroma, differences of PD-L1 positive macrophage during cycle 1 was also maintained in cell density (P = 0.02) and the percentage of PD-L1 positive cell (P = 0.06). Conclusion: TCZ+GN induced an increase of PD-L1 expression levels on tumor cell and macrophages. Citation Format: Kyoko Yamaguchi, Etsuko Fujii, Shuichi Mitsunaga, Noriaki Sawada, Masafumi Ikeda, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Mayu Makikawa, Kazunori Aoki, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Atsukiko Kato, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Increased PD-L1 expression levels were observed on both tumor cells and macrophages by tocilizumab plus gemcitabine/nab-paclitaxel treatment in gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6145.

Background: Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC) refractory to gemcitabine + nab-paclitaxel (GN) due to suppression of cancer associated fibroblast (CAF) and increase of drug infiltration in tumor. We sought to determine the safety, recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with GN-refractory MPC. Methods: This phase 1, dose finding trial was conducted following preclinical study of IL-6 signaling blockade in genetically engineered mouse model of pancreatic cancer (pGEMM) and patients-derived CAF, enrolled 10 patients with MPC that had progressed after GN. During the dose-finding part to determine dose-limiting toxicity (DLT), patients initially received TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. The subsequent dose-expansion part used the TCZ dose identified in the dose-finding study. Before Cycle 1 and at the end of Cycle 1, biopsy of liver metastasis was performed 3 to 5 hours after levofloxacin (LVXF) administration to measure LVFX infiltration in tumor using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). Results: In pGEMM tumor, mouse IL-6 receptor antibody plus GN led pathological response. The growth of CAFs from patients with pancreatic cancer was inhibited by TCZ. In this phase I study, no DLTs occurred and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events (TEAEs) occurred in 80% of patients (Grade ≥3 in 60%) with decreased neutrophil count (n=5, 7 events) being the most common TEAE. Disease control rate was 80·0% (95%CI: 44·4-97·5) and tumor reduction during cycle 1 was observed in 4 patients who were defined as responder. In paired-biopsied samples, decrease of phosphorylated (p) STAT3 expression in tumor was observed in 7 of 8 patients (88%). Responder-related biological activities were increase of cleaved PARP expression of tumor nuclei (P = 0.01), decrease of proliferative CAF (P = 0.08), and increase of LVFX infiltration in tumor (P = 0.04). Decrease of pSTAT3 expression (P = 0.02) was favor to increase of LVFX infiltration against increase of gamma-H2AX, an index of gemcitabine exposure (P = 0.20). Conclusion: TCZ+GN-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and gain of intratumoral drug infiltration in MPC. Citation Format: Shuichi Mitsunaga, Masafumi Ikeda, Kazunori Aoki, Kyoko Yamaguchi, Noriaki Sawada, Etsuko Fujii, Masanobu Nishidate, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Atsuhiko Kato, Mayu Makikawa, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT565.

Background: Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. The orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), was previously identified as expressed by mRNA in patients with multiple myeloma with only low expression detected in normal tissues, but confirmation of protein expression remained elusive. In this study, we determined the cell surface expression of GPRC5D on malignant and normal hematological cells. In addition, we evaluated the antitumor activity and mechanism of GPRC5D TRABs in in vitro and in vivo mouse models. Method & Results: We established specific monoclonal antibodies against human GPRC5D and identified its expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, did not find it at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. To investigate whether GPRC5D could be therapeutic target, we generated IgG-based anti-GPRC5D/CD3 bispecific T-cell-redirecting antibodies (GPRC5D TRABs). GPRC5D TRABs induced T cell activation and the killing of a wide variety of GPRC5D expressing tumor cells in vitro. In mouse models with reconstituted human immune cells, GPRC5D TRABs showed strong antitumor efficacy against GPRC5D-positive tumors through the activation of T cells. Conclusion: These findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy. Citation Format: Tatsushi Kodama, Yu Kochi, Waka Nakai, Hideaki Mizuno, Takeshi Baba, Kiyoshi Habu, Noriaki Sawada, Hiroyuki Tsunoda, Takahiro Shima, Kohta Miyawaki, Yoshikane Kikushige, Yasuo Mori, Toshihiro Miyamoto, Takahiro Maeda, Koichi Akashi. Anti -GPRC5D/CD3 T cell-redirecting bispecific antibody with potent in vitro and in vivo antitumor efficacy against multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1886.

Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. In this study, we identified G-protein–coupled receptor family C group 5 member D (GPRC5D) expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, not at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. In addition, we constructed IgG-based anti-GPRC5D/CD3 bispecific T-cell–redirecting antibodies (GPRC5D TRAB), which suppressed the tumor growth of GPRC5D-positive myeloma cells through the activation of T cells in vitro and in vivo in xenograft models. Collectively, these findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy.

Background: T cell-redirecting antibody (TRAB), which bispecifically binds to CD3 and tumor antigen, is a key player in next-generation cancer immunotherapy (CIT). Because TRAB can redirect T cells regardless of their TCR specificity, it is expected to be efficacious in immune checkpoint inhibitor-resistant tumors. The CD19-targeting bispecific T cell engager, blinatumomab, has been used for the treatment of blood cancers, and CEA-TCB and IMCgp100 have shown promising clinical efficacy in solid tumors as well. ERY974 is another promising TRAB targeting glypican-3 (GPC3) and a phase 1 study is in progress (Ishiguro et al., Sci Transl Med 2017). On the other hand, cytokine release syndrome (CRS) has been recognized as a common side effect of TRAB. Thus, mitigation of CRS is an urgent issue. Intra-patient step-up dosing regimens have been incorporated in clinical trials to reduce cytokine release, but the mechanism behind this phenomenon remains elusive. In this study, we pre-clinically explored the phenomenon and its mechanism by using ERY974. Method & Results: GPC3-expressing human cancer cells were incubated with human PBMCs and ERY974. The incubated PBMCs were harvested and mixed with newly prepared GPC3-expressing cancer cells and higher doses of ERY974. The pre-treated PBMCs showed reduced cytokine production compared to that without pre-treatment, while maintaining the same level of T cell-dependent cellular cytotoxicity (TDCC). Comprehensive gene expression analysis of the pre-treated PBMCs was also conducted. A murine cancer cell line expressing human GPC3 was established and implanted into immune competent mice. A mouse surrogate version of ERY974 (mERY974) was first administered at a low dose expected to elicit weak anti-tumor activity, and mice were then treated with a higher dose of mERY974. Anti-tumor activity was similar regardless of the pre-treatment, but plasma cytokine levels were shown to be reduced in the pre-treated mice upon administration of the higher dose of mERY974. Conclusion: Cytokine production following the high-dose ERY974 treatment was mitigated by the low-dose pre-treatment in vitro and in vivo. Cytotoxic activity did not decrease in this setting, thus demonstrating that the pre-treatment selectively suppressed only cytokine production. It is well known that repeated stimulation of TCR leads to T-cell exhaustion. The phenomenon we observed here can also be characterized as a sort of exhaustion, but is quite unconventional. Thus, we propose a new type of exhaustion and suggest calling it ‘Differential exhaustion on cytokine release (DECREASE)’. Management of CRS is critical for the clinical use of TRAB. A detailed analysis of the mechanism of DECREASE currently in progress, is expected to contribute to the development of desired dosing regimens for this new type of CIT agent. Citation Format: Mayumi Hoshino, Yuji Sano, Yasuko Kinoshita, Yumiko Azuma, Toshiaki Tsunenari, Yoko Kayukawa, Mizuho Noguchi, Takahiro Ishiguro, Shohei Kishishita, Noriaki Sawada, Mika Endo, Junichi Nezu. Differential exhaustion on cytokine release (DECREASE) by ERY974, a novel T-cell-redirecting antibody targeting glypican-3: A new type of T-cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2378.

Purpose Capecitabine has demonstrated high efficacy as first-line treatment for metastatic colorectal cancer (MCRC). Oxaliplatin shows synergy with fluorouracil (FU), with little toxicity overlap. The XELOX regimen (capecitabine plus oxaliplatin), established in a previous dose-finding study, should improve on infused oxaliplatin with FU and leucovorin (FOLFOX) regimens. The present studies further characterize efficacy and safety of the XELOX regimen. Patients and Methods The antitumor activity of XELOX was investigated in a colon cancer xenograft model. Patients with MCRC received first-line XELOX in 3-week treatment cycles: intravenous oxaliplatin 130 mg/m2 (day 1) followed by oral capecitabine 1,000 mg/m2 twice daily (day 1, evening, to day 15, morning). Results A preclinical study confirmed that capecitabine has supra-additive activity with oxaliplatin. In the clinical study, 53 of 96 patients (55%) achieved an objective response, and 30 (31%) experienced disease stabilization for ≥ 3 months following treatment. After 24 months' minimum follow-up, median time to disease progression (TTP) and median overall survival were 7.7 and 19.5 months, respectively. XELOX safety was predictable and similar to the FOLFOX4 regimen, except that myelosuppression was uncommon with XELOX (grade 3 or 4 neutropenia, 7%). Most adverse events were mild to moderate, the most common being acute sensory neuropathy (85%). Sixty-day, all-cause mortality was 2%. Conclusion XELOX is a highly effective first-line treatment for MCRC. Response rates, TTP, and overall survival are similar to those observed with FU/leucovorin/oxaliplatin combinations. XELOX provides a more convenient regimen, likely to be preferred by both patients and healthcare providers. Capecitabine has the potential to replace FU/LV in combination with oxaliplatin for MCRC.

An immediate obtainment of osseointegration would shortened the initial anchorage period of implant within bone and setting time of the final superstructure. Application of pulsing electromagnetic fields (PEMFs) as a method for increasing the rate of osseointegration has been considered. PEMFs have been demonstrated to promote the proliferation and differentiation of osteoblast-like clone MC3T3-E1 cells.Recently, the immunohistochemical staining technique having Brd U or PCNA has been used as markers to study the proliferation dynamics of S-phase cells or late G, to S-phase cells. In this study, cell proliferation was examined in the experimental group having PEMFs-stimulation and in the control group lacking stimulation by immunohistochemical staining technique using Brd U and PCNA monoclonal antibodies. Brd Ulabeling and PCNA- positive indices were respectively calculated and compared. Further, alkaline phosphatase activity was investigated as a marker for cell differentiation of MC3T3-E1 cells by using a histochemical staining technique.The results suggested that the Brd U-labeling index and PCNA-labeling index distinctly increased in the experimental group (P

We examined the efficacy of chemoendocrine therapy using capecitabine as a chemotherapeutic agent in premenopausal and postmenopausal models with estrogen receptor (ER)-positive human breast cancer xenografts. Tamoxifen and letrozole were used as endocrine therapeutic agents for premenopausal and postmenopausal models, respectively. The antitumor activity of capecitabine in combination was significantly superior to either monotherapy treatment in both premenopausal (p

Bacterial O 6 -alkylguanine-DNA alkyltransferase (AGT) removes alkyl group from O 6 -alkylguanine and O 4 -alkylthymine residues in DNA, both of which are considered to be DNA damages most related to the induction of cancer and/or mutation. The repair process involves alkyl-transfer of an O -alkyl group to the active site of the enzyme, where an SH-group of cysteine residue plays the role of alkyl acceptor. In order to elucidate the chemical characteristics of substrates for this enzyme, dealkylation rates of O 6 -alkyldeoxyguanosine, O 4 -alkylthymidine and related compounds were measured using an alkyltransfer system. Thiophenol-triethylamine system was employed as an alkyl acceptor and twenty-one O -alkyl compounds were tested. Dealkylation proceeded with pseudo first order kinetics. The half-life of O 6 -methyldeoxyguanosine (MedG) was 122 h and no remarkable dependence on N-9 substituents (H, CH 3 and deoxyribose) was observed. A compound lacking 2-NH 2 group underwent demethylation about three times faster than O 6 -methylguanines did, while, a compound lacking imidazole moiety underwent demethylation about 2.5 times more slowly. The half-life of O 4 -methylthymidine (MedT) was 38 h and no remarkable dependence on N-1 (H, CH 3 and deoxyribose) and C-5 (H and CH 3 ) substituents was observed. Deethylation proceeded much more slowly than demethylation. Substitution of selenophenol for thiophenol resulted in a 4.5 times faster MedG demethylation rate. Demethylation rates were moderately correlated with values for NMR chemical shift of CH 3 group, an indicator of electron density, although the correlation curves of a series of MedG and MedT derivatives were quite different. This result suggests that some different ratedetermining factors other than electron density are playing a role. These findings may be of help in resolving the details of the mechanisms of enzymic repair by bacterial and mammalian AGT.

We have evaluated the antitumor activity of XELOX (a combination therapy of capecitabine (Xeloda) and oxaliplatin) in human colorectal and gastric cancer xenograft models. In human colorectal cancer model CXF280, antitumor activity of the combination at two-thirds of the maximum tolerated dose (MTD) was superior to that of each monotherapy at MTD. Furthermore, in human colorectal cancer model COL-05-JCK and human gastric cancer xenograft model GXF 97, the combination also showed at least additive antitumor activity. In addition, toxicity was not augmented with the combination therapy in these three models. As demonstrated using ELISA or immunohistochemistry, oxaliplatin in xenograft model tumors up-regulated the level of thymidine phosphorylase (dThdPase), a key enzyme for the metabolism of capecitabine to 5-fluorouracil. These results suggest that oxaliplatin might potentiate the antitumor activity of capecitabine by up-regulating the tumor level of dThdPase. Based on these results, clinical trials of XELOX against colorectal and gastric cancers are warranted.

5'-deoxy-5-fluorouridine (5'-DFUR, Furtulon) is activated to 5-fluorouracil (5-FU) by thymidine phosphorylase (dThdPase) highly expressed in many types of tumors. In previous studies, we demonstrated that taxanes (paclitaxel or docetaxel) up-regulated the tumor levels of dThdPase and enhanced the efficacy of 5'-DFUR in human colon and mammary xenograft models. In the present study, combination therapy of 5'-DFUR with taxanes in human gastric cancer xenograft models also showed, at the least, additive anti-tumor activity without significant augmentation of toxicity. Furthermore, paclitaxel up-regulated dThdPase expression in the tumor tissues as confirmed with ELISA and immunohistochemistry. These results suggest taxanes would potentiate the efficacy of 5'-DFUR by up-regulating the tumor levels of dThdPase in gastric xenograft models. Clinical trials of 5'-DFUR in combination with taxane against gastric cancer are warranted.

To identify an orally available fluoropyrimidine having efficacy and safety profiles greatly improved over those of parenteral 5-fluorouracil (5-FU: 1), we designed a 5-FU prodrug that would pass intact through the intestinal mucisa and be sequentially converted to 5-FU by enzymes that are highly expressed in the human liver and then in tumors. Among various N4-substituted 5'-deoxy-5-fluorocytidine derivatives, a series of N4-alkoxycarbonyl derivatives were hydrolyzed to 5'-deoxy-5-fluorocytidine (5'-DFCR: 8) specifically by carboxylesterase, which exists preferentially in the liver in humans and monkeys. Particularly, derivatives having an N4-alkoxylcarbonyl moiety with a C4-C6 alkyl chain were the most susceptible to the human carboxylesterase. Those were then converted to 5'-deoxy-5-fluorouridine (5'-DFUR: 4) by cytidine deaminase highly expressed in the liver and solid tumors and finally to 5-FU by thymidine phosphorylase (dThdPase) preferentially located in tumors. When administered orally to monkeys, a derivative having the N4-alkoxylcarbonyl moiety with a C5 alkyl chain (capecitabine: 6) The highest AUC and Cmax for plasma 5'-DFUR. In tests with various human cancer xenograft models, capecitabine was more efficacious at wider dose ranges than either 5-FU or 5'-DFUR and was significantly less toxic to the intestinal tract than the others in monkeys.

Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the oral cytostatic drugs capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine, Xeloda(trade mark)) and its intermediate metabolite doxifluridine [5'-deoxy-5-fluorouridine (5'-dFUrd, Furtulon((R)))] to 5-fluorouracil (5-FUra) in tumors. In a previous study, we found that several cytostatics were able to up-regulate tumor levels of dThdPase in a human colon cancer xenograft model. In the present study, we confirmed that the administration of cytostatics used for breast cancer treatment, such as taxanes and cyclophosphamide (CPA), up-regulated the tumor level of dThdPase in mammary tumor models as well. Because the dThdPase up-regulation was observed even when CPA was given orally, we investigated further the usefulness of combination therapy with the 2 oral drugs, 5'-dFUrd/capecitabine and CPA in mammary tumor models. Daily oral administration of CPA up-regulated human dThdPase levels in the tumor tissue of mice bearing a human mammary tumor xenograft, MX-1, whereas in the small intestine and liver, it did not affect levels of pyrimidine nucleoside phosphorylases (PyNPase) including dThdPase and uridine phosphorylase. The preferential up-regulation of PyNPase activity in the tumor by CPA administration was also confirmed in mice bearing a syngeneic murine mammary adenocarcinoma, A755. In both models, combination therapy of 5'-dFUrd/capecitabine with CPA showed synergistic antitumor activity, without significant potentiation of toxicity. In contrast, treatment with CPA and either 5-FUra or UFT (a mixture of tegafur and uracil) in combination showed only additive activity. Our results suggest that CPA and capecitabine/5'-dFUrd, both available for oral administration, would be good partners, and that clinical trials with this drug combination against breast cancer are warranted.

Capecitabine (N4-pentyloxycarbonyl-5′-deoxy-5-fluorocytidine) is a novel oral fluoropyrimidine carbamate, which is converted to 5-fluorouracil (5-FU) selectively in tumours through a cascade of three enzymes. The present study investigated tissue localisation of the three enzymes in humans, which was helpful for us to design the compound. Carboxylesterase was almost exclusively located in the liver and hepatoma, but not in other tumours and normal tissue adjacent to the tumours. Cytidine (Cyd) deaminase was located in high concentrations in the liver and various types of solid tumours. Finally, thymidine phosphorylase (dThdPase) was also more concentrated in various types of tumour tissues than in normal tissues. These unique tissue localisation patterns enabled us to design capecitabine. Oral capecitabine would pass intact through the intestinal tract, but would be converted first by carboxylesterase to 5′-deoxy-5-fluorocytidine (5′-dFCyd) in the liver, then by Cyd deaminase to 5′-deoxy-5-fluorouridine (5′-dFUrd) in the liver and tumour tissues and finally by dThdPase to 5-FU in tumours. In cultures of human cancer cell lines, the highest level of cytotoxicity was shown by 5-FU itself, followed by 5′-dFUrd. Capecitabine and 5′-dFCyd had weak cytotoxic activity only at high concentrations. The cytotoxicity of the intermediate metabolites 5′-dFCyd and 5′-dFUrd was suppressed by inhibitors of Cyd deaminase and dThdPase, respectively, indicating that these metabolites become effective only after their conversion to 5-FU. Capecitabine, which is finally converted to 5-FU by dThdPase in tumours, should be much safer and more effective than 5-FU, and this was indeed the case in the HCT116 human colon cancer and the MX-1 breast cancer xenograft models.

Capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) is a novel fluoropyrimidine carbamate that is converted to 5-fluorouracil (5-FUra) by three enzymes located in the liver and tumors; the final step is the conversion of 5'-deoxy-5-fluorouridine (5'-dFUrd) to 5-FUra by thymidine phosphorylase in tumors. The present study compared the efficacy of capecitabine and 5-FUra at their maximum tolerated doses in CXF280, HCT116, COLO205, and WiDr human colon cancer xenograft models, and measured subsequent 5-FUra and 5'-dFUrd levels in tumors and in the plasma and muscle. Capecitabine was effective in the first three models, whereas 5-FUra was effective only in CXF280, which is a cell line highly susceptible to fluoropyrimidines. In the three susceptible models, 5-FUra AUCs in tumors after capecitabine administration were 210 to 303 nmol x hr/g, whereas those after 5-FUra administration were 8.54 to 13.1 nmol x hr/g. In addition, capecitabine gave higher levels of 5-FUra AUC in tumors than in plasma (114- to 209-fold higher) and muscle (21.6-fold higher), whereas 5-FUra was not selectively distributed to tumors. In the refractory model, WiDr, 5-FUra AUC in tumors after capecitabine administration was only 62.8 nmol x hr/g, although the level of the intermediate metabolite 5'-dFUrd was high (AUC: 695 nmol x hr/g). The ratio of 5-FUra/5'-dFUrd levels in the WiDr tumors was 0.09, which was 23.8-fold lower than that in the HCT116 tumors. The mechanism of resistance would be the inefficient conversion of 5'-dFUrd to 5-FUra by thymidine phosphorylase in tumors. Thus, capecitabine might show its high efficacy as a result of delivering high levels of 5-FUra selectively to the tumors.

Treatment of calf thymus deoxyribonucleic acid (DNA) in vitro with a methylating carcinogen, N-methyl-N-nitrosourea (MNU), in phosphate buffer (pH 7.2) resulted in formation of O6,7-dimethylguanine residues in DNA besides the well-known methylated DNA adducts, 7-methylguanine, O6-methylguanine and 3-methyladenine. The product ratio (%) of O6,7-dimethylguanine versus 7-methylguanine was 0.32 after one MNU treatment. The significance of formation of O6,7-dimethylguanine residues in DNA is discussed briefly in relation to the carcinogenicity of MNU.

This study investigated the adhesive strength of cyanoacrylate dental adhesive, 'F.H cement' after being thermal-cycled in order to certify its stability in water. The adhesive strength was compared using Zinc phosphate cement, Glass ionomer cement, Dental adhesive resin and F.H cement when an ivory die and a cast crown were adhered by these different cements. Results of the study indicated the following: 1. The adhesive strength after being thermal-cycled indicated that F.H cement had a greater strength than the other cements (Zinc phosphate cement and Glass ionomer cement) and was equal to that of the Dental adhesive resin. 2. F.H cement was also found to be the strongest in adhesive strength when different tapers were used.