Comprehensive analyses using next-generation sequencing and immunohistochemistry enable precise treatment in advanced gastric cancer.

BACKGROUND: In advanced gastric cancer (AGC), most clinical trials are designed on the basis of protein expression or gene amplification of specific genes. Recently, next-generation sequencing (NGS) allowed us to comprehensively profile the tumor gene status. This study aimed to elucidate the profiling between gene alterations and protein expression in AGC to aid in future clinical trials on AGC. PATIENTS AND METHODS: Formalin-fixed, paraffin-embedded tumor samples from 121 stage III/IV gastric cancer patients were examined for protein expression of tyrosine kinase receptors (RTKs; ERBB2, EGFR, c-MET, and FGFR2) using immunohistochemistry (IHC). Furthermore, 409 cancer-related genes were sequenced to detect mutations and copy number variations using NGS. RESULTS: Most ERBB2 overexpression (IHC 3+) cases (80.0%) had ERBB2 amplification and did not have other RTK amplification or oncogene mutations. However, one-fourth of MET overexpression cases (25.0%) had ERBB2 alterations. EGFR and FGFR2 overexpression cases had ERBB2 alterations or other gene alterations such as KRAS or PIK3CA. On the other hand, most of the four RTK amplification cases (88.2%) were mutually exclusive with each amplification. However, RTK amplification did not simply correlate with protein overexpression, whereas cases with RTK high-level amplification had protein overexpression and rarely showed other co-existing gene alterations. CONCLUSION: AGC involves a complicated arrangement of protein expression and gene alterations. Comprehensive analyses of NGS and IHC will be necessary to design the optimal therapy for treating the appropriate population of patients in future clinical trials.

Effect of a tunnel-structured ß-tricalcium phosphate graft material on periodontal regeneration: a pilot study in a canine one-wall intrabony defect model.

BACKGROUND AND OBJECTIVE: Tissue regeneration is affected by the porosity, chemical properties and geometric structure of graft materials. Regeneration of severe periodontal defects, such as one-wall intrabony defects, is difficult because of reduced tissue support, and bone grafts are commonly used in such cases. In the present study, a tunnel-structured ß-tricalcium phosphate (tunnel ß-TCP) graft material designed to stimulate bone formation was fabricated. The objective of this pilot study was to evaluate the effect of this graft material on periodontal regeneration in one-wall intrabony defects in dogs. MATERIAL AND METHODS: Six male beagle dogs were used in this study. First, the mandibular second and third incisors were extracted. Experimental surgery was performed 12 wk after tooth extraction. Bilateral 4 × 8 mm (width × depth) one-wall intrabony defects were created in the mesial side of the mandibular canines. At the experimental sites, the defects were filled with tunnel ß-TCP, whereas the control defects were left empty. Twelve weeks after surgery, qualitative and quantitative histological analyses were performed. RESULTS: There were no signs of clinical inflammation 12 wk after surgery. Coronal extension indicative of new bone formation was higher at the experimental sites than at the control sites, although the differences between both the sites in the newly formed cementum and connective tissue attachment were not significant. Newly formed periodontal ligament and cementum-like tissue were evident along the root surface at the experimental sites. The inner surface of the tunnels was partially resorbed and replaced with new bone. New blood vessels were observed inside the lumens of tunnel ß-TCP. CONCLUSION: Tunnel ß-TCP serves as a scaffold for new bone formation in one-wall intrabony defects.

Comprehensive clinical and molecular characterization of claudin 18.2 expression in advanced gastric or gastroesophageal junction cancer.

BACKGROUND: We conducted comprehensive clinical and molecular characterization of claudin 18.2 expression (CLDN18.2) in advanced gastric or gastroesophageal junction cancer (GC/GEJC). PATIENTS AND METHODS: Patients with advanced GC/GEJC who received systemic chemotherapy from October 2015 to December 2019 with available tumor specimens were analyzed. We evaluated clinicopathological features of CLDN18.2 expression with four molecular subtypes: mismatch repair deficient, Epstein-Barr virus-positive, human epidermal growth factor receptor 2-positive, and others. In addition, programmed death-ligand 1 (PD-L1) combined positive score (CPS), genomic alterations, and the expression of immune cell markers were assessed. Clinical outcomes of standard first- or second-line chemotherapy and subsequent anti-programmed cell death protein 1 (anti-PD-1) therapy were also investigated according to CLDN18.2 expression. RESULTS: Among 408 patients, CLDN18.2-positive (moderate-to-strong expression in ≥75%) was identified in 98 patients (24.0%) with almost equal distribution in the four molecular subtypes or CPS subgroups. CLDN18.2-positive was associated with Borrmann type 4, KRAS amplification, low CD16, and high CD68 expression. Overall survival with first-line chemotherapy was not significantly different between CLDN18.2-positive and -negative groups [median 18.4 versus 20.1 months; hazard ratio 1.26 (95% confidence interval 0.89-1.78); P = 0.191] regardless of stratification by PD-L1 CPS ≥5. Progression-free survival and objective response rates of first- and second-line chemotherapy, and anti-PD-1 therapy also showed no significant differences according to CLDN18.2 status. CONCLUSIONS: CLDN18.2 expression in advanced GC/GEJC was associated with some clinical and molecular features but had no impact on treatment outcomes with chemotherapy or checkpoint inhibition. CLDN18.2-positive also had no impact on overall survival. This information could be useful to interpret the results from currently ongoing clinical trials of CLDN18.2-targeted therapies for advanced GC/GEJC and to consider a treatment strategy for CLDN18.2-positive GC/GEJC.

Impact of the 12-gene recurrence score assay on deciding adjuvant chemotherapy for stage II and IIIA/B colon cancer: the SUNRISE-DI study.

BACKGROUND: Recent advances in adjuvant chemotherapy for early colon cancer have widened physicians' recommendations on the regimen and duration (3 or 6 months) of the treatment. We conducted this prospective study to evaluate whether the 12-gene recurrence score (12-RS) assay affected physicians' recommendations on adjuvant treatment selection. PATIENTS AND METHODS: Patients with stage IIIA/IIIB or stage II colon cancer were enrolled. After the patients discussed adjuvant treatment with their treating physicians, the physicians filled in the questionnaire before assay indicating the treatment recommendation. When the 12-RS assay results were available, the physicians again filled in the questionnaire after assay. The primary endpoint was the rate of change in treatment recommendations from before to after the assay, with a threshold rate of change being 20%. Patients with stage IIIA/B to II were enrolled in a ratio of 2 : 1. RESULTS: Overall, the treatment recommendations changed in 40% of cases after obtaining 12-RS assay results. Recommendations were changed in 45% (80/178; 95% confidence interval, 37% to 53%; P < 0.001) and 30% (29/97; 95% confidence interval, 21% to 40%; P < 0.001) of patients with stage IIIA/B and II colon cancer, respectively. Patients with stage IIIA/B cancer had significantly more change than those with stage II cancer (P = 0.0148). From before to after the 12-RS assay, the percentage of patients whose physicians reported being confident in their treatment recommendations significantly increased from 54% to 81% in stage IIIA/B (P < 0.001) and from 65% to 83% in stage II (P < 0.001). CONCLUSION: Our study confirmed the usefulness of the 12-RS assay in aiding the physician-patient decision-making process for tailoring adjuvant chemotherapy for stage IIIA/B colon cancer.

Bone tissue reaction of nano-hydroxyapatite/collagen composite at the early stage of implantation.

The purpose of this study was to develop a new biodegradable bone substitute materials consisting of synthesized nano-size hydroxyapatite (nano-HAp) and Type I biodegradable honeycomb collagen sponge (HCS) composites. Bone defects in rabbit mandibles were prepared by a drill, and the composites were implanted into the bone defects. The HCS only and the HCS/calcined hydroxyapatite (HAp) composite were used as comparative materials. The bone tissues reaction at the early stage within 3 weeks after implantaion was investigated histologically. Amounts of new bone formation were determined by NIH-image analysis software using the histological sections. The amounts of the new bone formation were largest in the nano HAp/HCS compared to the comparative materials. Within 2 weeks after implantation, the nano-HAp/HCS composite was more rapidly exchanged by new bone than the comparative materials. From these results it was considered that the nano-HAp/HCS composites can be used as an effective biodegradable bone substitutive material.

Rat maf related genes: specific expression in chondrocytes, lens and spinal cord.

maf is a family of oncogenes originally identified from avian oncogenic retrovirus, AS42, encoding a nuclear bZip transcription factor. We have isolated two maf related cDNA clones, maf-1 and maf-2, from a rat liver cDNA library. Comparison of the sequence homologies of the proteins encoded by maf-1 and maf-2 with those of c-maf and chicken mafB indicated that maf-1 and maf-2 are the rat homologues of mafB and c-maf, respectively. Both genes are expressed at low levels in a wide variety of rat tissues, including spleen, kidney, muscle and liver. Immunohistochemical studies and in situ hybridization analyses show that maf-1 and maf-2 are strongly expressed in the late stages of chondrocyte development in the femur epiphysis and the rib and limb cartilage of 15 day old (E15) embryo in rat. Cartilage cells, induced by subcutaneous implantation of bone morphogenic protein, also expressed maf-1 and maf-2. In situ hybridization analyses of E15 embryos show that both genes are expressed in the eye lens and the spinal cord as well as the cartilage. However, the expression patterns of maf-1 and maf-2 in lens and spinal cord are different.

Preferential adsorption of dentin and bone acidic proteins on the (100) face of hydroxyapatite crystals.

Interaction of bone and dentin proteins with minerals is an elementary step in the regulation of mineralization in these tissues. Adsorption of acidic non-collagenous proteins on hydroxyapatite was examined using fluorescence-labeled protein and synthetic hydroxyapatite. Phosphophoryn, bone Gla protein, osteonectin and bone small proteoglycan II were prepared and labeled with fluorescein. All of these proteins were adsorbed on hydroxyapatite with a dissociation constant on the order of 10(-7) M. The more acidic proteins had lesser binding capacities. Hydroxyapatite single crystals were incubated with labeled proteins and observed with a fluorescence microscope. Phosphophoryn and other acidic proteins were adsorbed preferentially on the (100) face of the crystal. This preferential adsorption of the acidic proteins may be responsible for the morphogenesis of biological hydroxyapatite.

The osteoblastic MC3T3-E1 cells synthesized C-terminal propeptide of type I collagen, which promoted cell-attachment of osteoblasts.

In this study, we purified C-terminal propeptide of type I collagen (PICP) from the conditioned medium of osteoblastic MC3T3-E1 cells by chromatographic and Agarose gel extraction procedures. PICP was confirmed to be present in bone by Western blotting using a specific antibody, and was proved to be synthesized by osteoblasts with metabolic labeling. PICP promoted cell-attachment of osteoblastic MC3T3-E1 cells. We conclude that PICP is synthesized by osteoblasts and stored in bone, and that it plays a role in the maintenance of bone cells on bone matrix.

Proteochondroitin sulfate synthesized in cartilages induced in vivo and in vitro by bone matrix gelatin.

Implanted allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [35S]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting component had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan component obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.

Acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals.

Osteonectin, an acidic noncollagenous protein of bone and dentin, has affinity to hydroxyapatite crystals. Binding sites to hydroxyapatite of this protein were determined by a proteolytic experiment and an in vitro binding experiment using synthetic peptide analogues. Osteonectin was adsorbed on hydroxyapatite crystals and digested with trypsin. A peptide was left adsorbed on the crystal even after the digestion. The peptide was identified as an amino terminal peptide containing glutamic acid-rich sequences, which have been assumed to be possible hydroxyapatite-binding sites. Poly glutamic acid sequences were synthesized as models of the binding sites. Glu6 peptide was bound to the hydroxyapatite with a dissociation constant of 2.4 microM. Peptides containing fewer glutamic acids had lower affinity to the crystal. Effects of these peptides on in vitro mineralization were examined by a gel system in microtiter plates. The Glu6 peptide had a positive effect on the mineralization in this system, whereas Asp6 peptide had a negative effect. These effects indicate the presence of an interaction between these peptides and mineral crystals.

A high-density rice genetic linkage map with 2275 markers using a single F2 population.

A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using > 130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.

Growth factor-loaded scaffolds for bone engineering.

The objective of the study presented here was to investigate the bone inductive properties as well as release kinetics of rhTGF-beta1- and rhBMP-2-loaded Ti-fiber mesh and CaP cement scaffolds. Therefore, Ti-fiber mesh and porous CaP cement scaffolds were provided with these growth factors and inserted in subcutaneous and cranial implant locations in rats and rabbits. In vitro, a rapid release of rhTGF-beta1 was observed during the first 2 h of the Ti-fiber mesh scaffolds. During this time, more than 50% of the total dose of rhTGF-beta1 was released. Following this initial peak, a decline in the level of rhTGF-beta1 occurred. After 1 week, the entire theoretical initial dose was observed to have been released. This in contrast to the rhTGF-beta1 and rhBMP-2 release of the porous CaP cement scaffolds. Here, no substantial initial burst release was observed. The scaffolds showed an initial release of about 1% after 1 day, followed by an additional marginal release after 1 week. Histological analysis revealed excellent osteoconductive properties of non-loaded Ca-P material. Inside non-loaded Ti-mesh fiber scaffolds, also bone ingrowth occurred. Quantification of the bone ingrowth showed that bone formation was increased significantly in all scaffold materials by administration of rhTGF-beta1 and rhBMP-2. Consequently, we conclude that the release kinetics of growth factors from porous CaP cement differs from other scaffold materials, like metals and polymers. Nevertheless, orthotopic bone formation in a rabbit cranial defect model was stimulated in rhTGF-beta1- and rhBMP-2-loaded CaP cement and Ti-fiber mesh scaffolds compared with non-loaded implants.

Perturbation of BMP signaling in somitogenesis resulted in vertebral and rib malformations in the axial skeletal formation.

Axial skeletons such as vertebrae, ribs, and scapulae develop from the embryonic somitic mesoderm through interactions with neural tube/notochord and skin ectoderm. Bone morphogenetic proteins (BMPs) seem to play important roles in these tissue interactions; however, the relationship between BMP signaling and the early development of axial skeletons is poorly understood. In this report, we investigated possible roles of BMP signaling in axial skeletal formation. First, we describe the expression patterns of BMP4 and type I receptors for BMP during somitogenesis in chick embryos based on whole mount in situ hybridization. Next, the effects of BMP on axial skeletal morphogenesis were investigated by implantation of BMP proteins into the dorsal mesoderm at the time of somitogenesis. Transcripts for both BMP4 ligand and its receptors are expressed in the dorsal ectoderm and mesoderm. Implantation of BMP4 and BMP2 into the dorsal regions of embryos result in subsequent anomalies of vertebrae, ribs, and scapulae. The effects of BMP implantation on the skeleton are shown to be dependent upon the somitic stage. Vertebral anomalies are restricted to the dorsolateral elements of the vertebrae and specifically observed after BMP implantation into embryonic day 2 (E2) embryos, but not E3 embryos. These results indicate that implantation of BMP into the dorsal part of embryos where endogenous BMP ligand and BMP receptors are expressed perturbs BMP signaling and causes axial skeletal malformations. The findings presented here suggest that BMP signaling may be involved in the early developmental process of the axial skeleton.

Attachment of osteoblastic cells to hydroxyapatite crystals by a synthetic peptide (Glu7-Pro-Arg-Gly-Asp-Thr) containing two functional sequences of bone sialoprotein.

We investigated activity of bone sialoprotein (BSP) to mediate attachment of cells to hydroxyapatite using a model peptide, Glu7-Pro-Arg-Gly-Asp-Thr, which contains a putative hydroxyapatite-binding site (poly-Glu) and a cell-attachment site. The peptide has affinity to hydroxyapatite with a dissociation constant of 13.5 microM. The peptide affected in vitro mineralization in a gel system, indicating interaction between this peptide and calcium phosphate. The osteoblastic cell line MC3T3-E1 was incubated with hydroxyapatite powder coated with the peptide or proteins. Attachment of the cells was observed on the powder coated with BSP, but not on the powder coated with serum albumin. The cells were attached to the powder coated with the peptide. The cells were flattened on the powder, and pseudopods developed. The attachment of the cells was inhibited by an excessive amount of Gly-Arg-Gly-Asp-Ser peptide. In conclusion, BSP mediated attachment of osteoblastic cells to hydroxyapatite, and this activity could be accomplished only by the poly-Glu sequence and the Arg-Gly-Asp sequence.

Carboxyl-terminal propeptide of type I collagen (c-propeptide) modulates the action of TGF-beta on MC3T3-E1 osteoblastic cells.

Previously we found that the carboxyl-terminal propeptide of type I collagen (c-propeptide) is a major secretory protein of MC3T3-E1 osteoblastic cells. In this study, we found that c-propeptide suppresses collagen synthesis and alkaline phosphatase activity of MC3T3-E1 osteoblastic cells at the early-differentiated stage in a dose dependent manner. Mature osteoblasts did not respond to c-propeptide. These findings imply that c-propeptide modulates the function of osteoblasts at an early differentiation stage. Transforming growth factor-beta (TGF-beta) is stored in bone and released from bone matrix after the resorption by osteoclasts. We investigated the effect of c-propeptide on the action of TGF-beta, and found that it enhanced the effect of TGF-beta. We conclude that c-propeptide is a physiological modulator of TGF-beta in bone metabolism.

Promotion of the osteogenetic activity of recombinant human bone morphogenetic protein by prostaglandin E1.

We investigated the promotive effect of prostaglandin (PG) E1 on the osteogenetic activity of bone morphogenetic protein (BMP) using porous chemically synthesized hydroxyapatite ceramic (HAP) pellets as the carrier. After treating the pellets with recombinant human (rh) BMP-2 with or without PGE1, both of which were used at two different concentrations, they were inserted beneath the cranial periosteum of a rabbit. The degree of osteogenesis and osteoconductivity was then examined histopathologically as well as by means of an image-analyzing procedure. Results showed that there was extensive bone formation around the pellets as early as 3 weeks after insertion in the group, which received pellets treated with a relatively large amount of rhBMP alone as well as in the group receiving pellets treated with a small amount of rhBMP combined with PGE1. In the later group, the extent of bone formation was dose-dependent. Subsequent osteogenesis within the pores of the pellets in these groups slowly progressed over time, and by 9 weeks after the insertion, most of the pellet pores had filled with newly generated bone. In addition, the groups which received pellets treated with PGE1 alone also showed osteogenesis as demonstrated by osteoconduction, which was not observed in the group that received the phosphate buffered saline (PBS) treated pellets. The group that received pellets treated with a small amount of rhBMP plus a high concentration of PGE1 exhibited significantly greater bone induction than the group which received the pellets treated with a small amount rhBMP alone, and there was no statistically significant difference between the former group and the group that received a high rhBMP dose. These results clearly indicated that PGE1 has a strong and dose-dependent promotive effect on the osteogenetic activity of rhBMP and that it also promotes osteoconduction, even when used alone. This suggests that enormous reductions can be made in the amount of rhBMP administered when PGE1 is used in conjunction with porous HAP. This should prove to be very advantageous and practical in the clinical application of these materials.

Osteogenesis by bone marrow stromal cells maintained on type I collagen matrix gels in vivo.

In this study, we demonstrated that bone marrow stromal cells maintained on type I collagen matrix induced bone in vivo. The formed bone contained bone marrow, and the process of bone formation occurred without cartilage formation. Bone marrow stromal cells differentiated into osteoblasts on type I collagen matrix in vitro, but types II, III, and V collagens did not possess this activity. These findings imply that type I collagen matrix offers a suitable environment for the induction of osteoblastic differentiation in vitro and osteogenesis in vivo.

Mineral induction by immobilized phosphoproteins.

Dentin phosphoproteins are thought to have a primary role in the deposition of mineral on the collagen of dentin. In this study we determined the type of binding between collagen and phosphoproteins necessary for mineral formation onto collagen fibrils and whether the phosphate esters are required. Bovine dentin phosphophoryn or phosvitin from egg yolk were immobilized on reconstituted skin type I collagen fibrils by adsorption or by covalent cross-linking. In some samples the ester phosphate was removed from the covalently cross-linked phosphoproteins by treatment with acid phosphatase. All samples were incubated at 37 degrees C in metastable solutions that do not spontaneously precipitate. Reconstituted collagen fibrils alone did not induce mineral formation. The phosphoproteins adsorbed to the collagen fibrils desorbed when the mineralization medium was added, and mineral was not induced. The mineral induced by the cross-linked phosphoproteins was apatite, and the crystals were confined to the surface of the collagen fibrils. With decreasing medium saturation the time required for mineral induction increased. The interfacial tensions calculated for apatite formation by either phosphoprotein cross-linked to collagen were about the same as that for phosphatidic acid liposomes and hydroxyapatite. This similarity in values indicates that the nucleation potential of these highly phosphorylated surfaces is about the same. It is concluded that phosphoproteins must be irreversibly bound to collagen fibrils for the mineralization of the collagen network in solutions that do not spontaneously precipitate. The phosphate esters of phosphoproteins are required for mineral induction, and the carboxylate groups are not sufficient.

Properties and cytotoxicity of water soluble Na2O-CaO-P2O5 glasses.

Various compositions of Na2OCaO-P2O5 glasses are prepared to estimate glass formation, dissolution properties and cytotoxicity. In the wide composition range of 40 mol% of P2O5 or more, clear glass samples were obtained. The estimated glass forming region was consistent with other ternary phosphate glass systems. The glass transition temperatures and crystallization temperatures decreased with increasing P2O5 content and increased with CaO content. Dissolution properties in distilled water and simulated body fluid (SBF) were measured. In distilled water, CaO free glasses showed extremely fast dissolution. The dissolution rate decreased with increasing CaO content and decreasing P2O5 content. This composition effect results from cross-link formation between the non-bridging oxygens of two different chains by Ca2+ ions which improves the phosphate network strength. In SBF, the dissolution rate followed a similar trend, but glass dissolution was suppressed. This suppression occurred due to the existence of soluble species of glass such as Na+, Ca2+ and HPO(2-)4. The cytotoxicity decreased with increasing CaO content and with decreasing PO2.5 content. This was the result of a change in pH and ion concentration in the medium.