Status and prognostic value of immunological biomarkers of breast cancer.

The immune response to cancer serves an important role in disease progression and patient prognosis. For triple-negative breast cancer showing aggressive behavior, immunotherapy has a good efficacy because of the potent immunogenicity of this type of cancer. However, the dominant subtype, luminal human epidermal growth factor receptor-2 (HER2)-negative breast cancer, is less immunogenic. To determine whether luminal HER2-negative cancer reacts to the anticancer immune response, the present study analyzed the status and prognostic value of the principal immunological biomarkers of breast cancer, including tumor-infiltrating lymphocytes (TILs), CD8+ T lymphocytes, the major histocompatibility complex and programmed cell death ligand-1 (PD-L1). The biomarkers were compared between patients with luminal HER2-negative breast cancer and those with immunogenic subtypes including triple-negative and HER2-overexpressed breast cancer. A total of 71 patients with primary breast cancer were classified into the immunogenic non-luminal (n=23) and less immunogenic luminal HER2-negative groups (n=48) based on immunogenicity. In the luminal HER2-negative group, compared with patients with low TIL levels, those with high TIL levels were at an advanced stage of cancer (P=0.024) and showed worse relapse-free survival (P=0.057); however, the remaining biomarkers exhibited no association with cancer progression or prognosis. In the non-luminal group, patients with high TIL levels showed significantly better RFS than those with low TIL levels (P=0.014). Compared with non-luminal patients negative for PD-L1, those positive for PD-L1 exhibited better overall survival (P=0.064). Notably, TIL status was found to exhibit contrasting prognostic predictions based on immunogenicity. In conclusion, TILs are a strong candidate for prognostic prediction in breast cancer, regardless of the subtype. PD-L1 is a potential candidate for prognostic prediction in immunogenic breast cancers, but not in the luminal HER2-negative subtype.

Ethylene treatment of "Maekawa-Jiro" persimmon affects peel characteristics and consequently, enables boil-peeling.

In a previous study, we reported that ethylene treatment facilitated boil-peeling in persimmons and in several other fruits; however, the mechanism underlying the facilitating effect of ethylene was not examined in detail. Thus, in this study, we investigated the effect of ethylene treatment on the peel characteristics of persimmons, that facilitated boil-peeling, using chemical, genomic, and histochemistry analyses. The results of the study showed that the ethylene-related genes, DK-ACS1 and DK-ACO2, and the pectinase-active gene DKPG were not expressed, even though a minor increase in ethylene generation was observed after ethylene treatment. Conversely, significant accumulation of toluidine blue O and ruthenium red dyes were observed in the sarcocarp and exocarp of the fruits, indicating an increase in the quantity of polysaccharides, including pectic substances, at the site. The results also indicate that the increased cellulase activity observed in the pericarp of the fruits may be due to the aging of the fruits, and not necessarily as a result of ethylene treatment. Furthermore, ethylene treatment increased the quantity of polysaccharides, including pectic substances, directly below the pericarp, which caused the dissolution of the site, resulting in peeling. This study provides new insights on the effect of ethylene on boil-peeling in persimmons and provides a foundation for future research studying the effect of heat treatment in the peeling of fruits or tomato.

Preexisting diabetes mellitus had no effect on the no-observed-adverse-effect-level of acetaminophen in rats.

Information on the safety of chemical substances in patients with various preexisting conditions remains limited. Acetaminophen was added to the basal diet at 0, 80, 253, 800, 2530, or 8000 ppm and administered to type 2 diabetes mellitus rats (GK/Jcl) and the control male rats (Wistar) for 13 weeks. Both strains treated with 8000 ppm acetaminophen (561.4 and 567.7 mg/kg body weight/day, GK/Jcl and Wistar rats, respectively) showed decreased levels of red blood cell counts, blood urea nitrogen, creatinine, and total bilirubin compared to those of non-treated rats. Treatment with 8000 ppm of acetaminophen reduced the blood glucose and hemoglobin A1c levels of GK/Jcl rats. An increase in the relative weights of the kidneys and liver, and a decrease in the weight of the salivary glands were observed in both GK/Jcl and Wistar rats treated with 8000 ppm acetaminophen relative to those of non-treated control rats. Microscopically, both strains treated with 2530 (174.3 and 164.2 mg/kg body weight/day, GK/Jcl and Wistar rats, respectively) or 8000 ppm acetaminophen showed hepatocellular hypertrophy and degenerative lesions in the salivary glands, whereas similar lesions were not observed in non-treated rats. In conclusion, the no-observed-adverse-effect-level of acetaminophen was 800 ppm in both diabetic and control rats.

Lung proliferative lesion-promoting effects of left pulmonary ligation in A/J female mice.

This present study was conducted in an attempt to examine proliferative lesion-promoting effect in the lung by compensatory lung growth after left pulmonary ligation. To examine a strong proliferative lesion-promoting effect in the lung, the effects of left pulmonary ligation on lung proliferative lesions induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were examined for 12 weeks. The number of proliferative lesions induced by NNK in the right lung after left pulmonary ligation increased significantly after 12 weeks, indicated by an increase in the weight of the right lung. In addition, several messenger RNA (mRNA) markers, including insulin growth factor 1, were highly expressed in the right lung on the seventh day after left ligation. These experiments demonstrated the clear proliferative lesion-promoting effects of pulmonary ligation on the induction of the expression of mRNAs related to the cell cycle, cell division and mitosis. However, the proliferative lesion-promoting effects were not strong enough to allow a shortened experimental period for the establishment of the lung bioassay model. The results also indicated the necessity to pay attention to the possibility of a recurrence of lung cancer in the residual lung after resection in humans.

Ethylene facilitates boil-peeling in fruits.

Boil-peeling is a common method of cooking or processing some horticultural crops. While boil-peeling is possible in some horticultural crops, a comprehensive list of crops for which boil-peeling is possible does not exist. According to a previous study, ethylene facilitates boil-peeling of kiwifruits. Thus, we studied the effect of ethylene treatment on boil-peeling in the kiwifruit variety "Rainbow red." We found that with increasing ethylene concentration in the fruits, boil-peeling success of kiwifruits increased. In the no-ethylene treatment, flesh firmness of the fruits decreased and boil-peeling could not be carried out successfully. Thus, it was clear that ethylene facilitates boil-peeling in kiwifruit. Furthermore, boil-peeling was possible after ethylene treatment in persimmon and Japanese pear, which had proved to be impossible so far. Kiwifruits, persimmon, and Japanese pear were classified as climacteric fruits that react with high ethylene sensitivity. Thus, ethylene may facilitate boil-peeling in climacteric fruits. This finding can possibly suggest new application for ethylene during fruit processing or in processed fruits.

[Efficacy of TS-1 as a Late-Line Treatment for Metastatic Breast Cancer].

We report 4 female patients with metastatic breast cancer who were administered TS-1 as a late-line treatment and showed favorable outcomes. Their average age was 66.3. The patients, all of whom had undergone prior treatment with both anthracyclines and taxanes, showed intrinsic Luminal A or B subtypes. After administration of TS-1 in the lines of 2 to 9 in metastatic settings, all patients showed a long progression-free survival with a favorable quality of life.

Development and Clinical Trials of Nucleic Acid Medicines for Pancreatic Cancer Treatment.

Approximately 30% of pancreatic cancer patients harbor targetable mutations. However, there has been no therapy targeting these molecules clinically. Nucleic acid medicines show high specificity and can target RNAs. Nucleic acid medicine is expected to be the next-generation treatment next to small molecules and antibodies. There are several kinds of nucleic acid drugs, including antisense oligonucleotides, small interfering RNAs, microRNAs, aptamers, decoys, and CpG oligodeoxynucleotides. In this review, we provide an update on current research of nucleic acid-based therapies. Despite the challenging obstacles, we hope that nucleic acid drugs will have a significant impact on the treatment of pancreatic cancer. The combination of genetic diagnosis using next generation sequencing and targeted therapy may provide effective precision medicine for pancreatic cancer patients.

[Utility of Prophylactic Administration of Pegfilgrastim in Breast Cancer Chemotherapy].

Febrile neutropenia(FN)is a frequent adverse event observed in cancer patients undergoing chemotherapy that may cause life-threatening infections. However, reducing the dose of anti-cancer drugs for breast cancer in adjuvant settings to prevent FN has been reported to adversely affect patient survival. Therefore, it is important to administer therapeutic agents as per their prescheduled regimens without delays or reductions in the dosage. From April 2015 to September 2017, pegfilgrastim was administered to 24 patients with breast cancer(primary prevention in 11 patients and secondary prevention in 13 patients)to prevent FN during chemotherapy in either adjuvant or metastatic settings. We were able to reduce the incidence of FN through prophylactic administration of pegfilgrastim without encountering serious adverse events. The inclusion of pegfilgrastim is considered essential for the safe administration of chemotherapy according to a preplanned schedule. Here, we discuss the indications, efficacy, and safety of the drug.

Characteristics of surfactant proteins in tumorigenic and inflammatory lung lesions in rodents.

Surfactant proteins (SPs) are essential for the proper structure and respiratory function of the lungs. There are four subtypes of SPs: SP-A, SP-B, SP-C, and SP-D. The expectorant drug ambroxol hydrochloride is clinically used to stimulate pulmonary surfactant and airway serous secretion. In addition, previous studies showed that ambroxol regulated SP production and attenuated pulmonary inflammation, with ambroxol hydrochloride being found to suppress quartz-induced lung inflammation via stimulation of pulmonary surfactant and airway serous secretion. In this study, we investigated the expression of SP-A, SP-B, SP-C, and SP-D in neoplastic and inflammatory lung lesions in rodents, as well as their possible application as potential markers for diagnostic purposes. SP-B and SP-C showed strong expression in lung hyperplasia and adenoma, whereas SP-A and SP-D were expressed in the mucus or exudates of inflammatory alveoli. Rodent tumorigenic hyperplasic tissues induced by various carcinogens were positive for napsin A, an aspartic proteinase involved in the maturation of SP-B; this indicated a focal increase in type II pneumocytes in the lungs. Therefore, high expression of napsin A in the alveolar walls may serve as a useful marker for prediction of the tumorigenic potential of lung hyperplasia in rodents.

Effects of the expectorant drug ambroxol hydrochloride on chemically induced lung inflammatory and neoplastic lesions in rodents.

Ambroxol hydrochloride (AH) is an expectorant drug used to stimulate pulmonary surfactant and serous airway secretion. Surfactant proteins (SPs) are essential for maintaining respiratory structure and function, although SP expression has also been reported in lung inflammatory and proliferative lesions. To determine whether AH exerts modulatory effects on these lung lesions, we examined its effects on pleural thickening induced by intrathoracic administration of dipotassium titanate (TISMO) in A/JJmsSlc (A/J) mice. We also analyzed the modulatory effects of AH on neoplastic lung lesions induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice and by N-nitrosobis (2-hydroxypropyl) amine (DHPN) in F344/DuCrlCrj (F344) rats. A/J mice treated with TISMO showed decreased body weight, increased white blood cell (WBC) counts, and pleural thickening caused by pleuritis and poor general condition. However, A/J mice treated with TISMO + 120 ppm showed significant recovery of body weight and WBC counts to the same levels as those of A/J mice not treated with TISMO, although no significant differences were observed in histopathological changes including the immunohistopathological expression of IL-1ß in the lung and maximum pleural thickness regardless of AH treatment. In the NNK and DHPN experiments, no significant differences in body weight, hematology, plasma biochemistry, and histopathological changes were associated with AH concentration. These results suggest that AH potentially exerts anti-inflammatory effects but does not have a direct suppressive effect on lung tumorigenesis in rodents.

γH2AX is immunohistochemically detectable until 7 days after exposure of N-bis (2-hydroxypropyl) nitrosamine (DHPN) in rat lung carcinogenesis.

It is known that γH2AX, which is formed when there is a double-strand break in DNA, can act as a sensitive marker of genomic instability. In this experiment, the time-course manner of the expression of γH2AX in the lung was examined in the early phase after treatment with a lung carcinogen, N-bis (2-hydroxypropyl) nitrosamine (DHPN). The expression of γH2AX is expected to be one of the useful markers for lung carcinogenesis in early stages. Rats were separated into 10 groups of 5 rats. The DHPN groups were administered 0.1% DHPN in drinking tap water for two weeks, while the control group received drinking tap water. At 0, 1, 3, 7, and 14 days after finishing DHPN treatment, one group each from the DHPN and control groups was sacrificed. The removed lung tissues were examined for immunostaining of γH2AX and PCNA, and positive cells were counted. The γH2AX levels of the DHPN-treated groups were found to be increased significantly at 0, 1, 3, and 7 days (4.4 ± 1.4, 5.1 ± 2.7, 3.3 ± 1.0, and 4.1 ± 1.3%, respectively), and they dropped significantly on day 14 (1.1 ± 0.4%). The experiment showed that the γH2AX-positive score could be effectively measured for up to 7 days after exposure, as a significance difference was observed between the treated group and the control group. It can be deduced that γH2AX is an effective marker for DHPN-induced double-strand breaks in pulmonary epithelial cells.

Validating the use of napsin A as a marker for identifying tumorigenic potential of lung bronchiolo-alveolar hyperplasia in rodents.

There are two types of bronchiolo-alveolar hyperplasia (hyperplasia) in rodent lungs. The first is "inflammatory hyperplasia" that retains its ability to revert to normal epithelia upon removal of the stimulating insult. The second is "latent tumorigenic hyperplasia", which is irreversible and causes independent preneoplastic lesions that can progress to bronchiolo-alveolar adenocarcinoma. Previously, lung samples with hyperplastic lesions were obtained from rats exposed to N-bis (2-hydroxypropyl) nitrosamine (DHPN) and fine particles (e.g. quartz), and 19 specific markers were examined immunohistochemically to identify latent tumorigenic hyperplasia. In the cytoplasm of the cells that make up the alveolar wall, we found that napsin A was weakly expressed in the inflammatory hyperplastic lesions, and was strongly expressed in the latent tumorigenic hyperplastic lesions induced by DHPN. To validate the possibility that napsin A may serve as a tumorigenic hyperplastic marker, additional experiments were performed with rats and mice. Latent tumorigenic hyperplasia induced by various carcinogens were positive for napsin A, similar to hyperplasia induced by DHPN. Thus, high expression of napsin A in alveolar walls may serve as a useful marker for detecting the tumorigenic potential of lung hyperplasia in rodents.

Suppressive effects of the expectorant drug ambroxol hydrochloride on quartz-induced lung inflammation in F344 rats.

Surfactant proteins (SPs) are essential to respiratory structure and function. The expectorant drug ambroxol hydrochloride is clinically prescribed to stimulate pulmonary surfactant and airway serous secretion. Therefore, ambroxol hydrochloride may affect SP production and pulmonary inflammation. Lung toxicity of fine particles of various materials has been examined previously in our in vivo bioassay using the intratracheal (i.t.) instillation approach. In the present study, we evaluated modulatory effects of ambroxol hydrochloride on quartz-induced lung inflammation in F344 rats. Male 6-week-old F344 rats were exposed by i.t. instillation to 2 mg of quartz particles suspended in 0.2 mL of saline. Ambroxol hydrochloride was administered at 0, 12, and 120 ppm in rat basal diet for 28 days, and then formalin-fixed paraffin-embedded lung, liver, and kidney samples were prepared. No changes in general condition, body and organ weights, or food consumption upon exposure to quartz were noted. The mean ambroxol intake in rats of the 12 ppm group was comparable to the human conventional dose. Histopathology of lung lesions was evaluated, and the degree of inflammation was scored. At 120 ppm, ambroxol hydrochloride significantly decreased individual lung inflammation scores for pulmonary edema and lymph follicle proliferation around the bronchiole, as well as the total inflammation score, in quartz-treated rats. Expression of SP-C in the type II alveolar cells and macrophages was greater in inflammatory lesions than in non-inflamed areas. Ambroxol treatment did not affect expression of SP-B and SP-C. In conclusion, we demonstrated that ambroxol hydrochloride relieves quartz-induced lung inflammation.

Chronic mesothelial reaction and toxicity of potassium octatitanate fibers in the pleural cavity in mice and F344 rats.

Fiber-shaped particles of potassium octatitanate (tradename TISMO; chemical formula K2 O·6TiO2 ), which are morphologically similar to asbestos particles, were shown to induce severe proliferative reactions in the pleural mesothelium in a previous experiment carried out over 21 weeks. The present study aims to determine whether these fibers induce malignant mesotheliomas in rodents, and to examine chronic toxicity induced. Additionally, we investigated the specific differences observable between the biological responses to the direct infusion of the fibers alone into the pleural cavity and those induced by the co-administration of the fibers with a known carcinogen. To detect the induction of malignant pleural mesotheliomas, two experiments were undertaken. In Experiment 1, four strains of mice, A/J, C3H, ICR, and C57BL, were examined for 52 weeks after experimental treatment with TISMO. In Experiment 2, the F344 rats were treated with TISMO alone, the lung carcinogen N-bis (2-hydroxypropyl) nitrosamine (DHPN) alone, both TISMO and DHPN, or left untreated and were then examined for 52 weeks. In this experiment, malignant lesion induction was expected in the co-administration group. TISMO fibers were observed in the alveoli, indicating penetration through the visceral pleura in mice and rats. The histopathological detection of TISMO fibers in the liver and kidneys of mice and rats indicated migration of the fibers out of the pleural cavity. Atypical mesothelial cells with severe pleural proliferation were observed, but malignant mesotheliomas were not detected. Among the rats, there were no observed malignant alterations in the mesothelium induced by DHPN-TISMO co-administration.

Long-Term Chronic Toxicity and Mesothelial Cell Reactions Induced by Potassium Octatitanate Fibers (TISMO) in the Left Thoracic Cavity in A/J Female Mice.

The present study was conducted to examine the chronic effects of potassium octatitanate fibers (trade name TISMO; chemical formula K2O·6TiO2) on the mouse lung and thoracic cavity. This method of infusion was employed to examine the direct effects of the fibers to the pleura. In the present study, 52- and 65-week experiments were employed to examine the long-term chronic effects after infusion of fiber-shaped TISMO into the thoracic cavities of A/J mice. Following this infusion, TISMO fibers were observed in the alveoli, indicating penetration through the visceral pleura. The additional histopathological detection of TISMO fibers in the liver, spleen, kidneys, ovary, heart, bone marrow, and brain of TISMO-infused mice indicated migration of the fibers out from the thoracic cavity. Atypical mesothelial cells with severe pleural proliferation were observed, but malignant mesotheliomas were not detected. This study demonstrated that intrathoracic infusion of TISMO fiber did not cause malignant mesothelioma but did cause severe chronic inflammation and proliferation of pleural mesothelial cells.

Rat strain differences in levels and effects of chronic inflammation due to intratracheal instillation of quartz on lung tumorigenesis induced by DHPN.

Chronic inflammatory effects of single intratracheal instillation (i.t.) of quartz on rat lung tumorigenesis were examined using 4 different animal models. At first, in order to determine an appropriate dose of quartz i.t. to promote lung tumorigenesis, F344 male rats were administrated single 0, 0.5, 1, 2 or 4 mg quartz/rat after initiation by N-bis(2-hydroxypropyl) nitrosamine (DHPN). Further studies were performed to examine strain differences of the effects of chronic inflammation caused by quartz i.t. in 3 strains of rat, i.e. F344, Wistar-Hannover and SD. Each was instilled with 2mg quartz/rat after DHPN administration and sacrificed in week 24. In addition, strain differences in generation of inflammation were determined at days 1 and 28. Finally, for determination of long-term effects period, F344 and Wistar-Hannover rats were similarly treated, but the experiment was terminated at week 52. In F344 rats, the tumor areas in DHPN treated groups showed a tendency to increase along with the dose of quartz. F344 rats demonstrated the highest and Wistar-Hannover rats the lowest sensitivity to quartz in acute and chronic phases in the 3 strains. In 52 week, in F344 rats, the multiplicity of tumors and the serum concentration of IL-6 in the group treated with DHPN and quartz were significantly increased. The present experiments indicated that chronic inflammation due to quartz instillation exerted promoting effects on lung carcinogenesis in F344, SD and Wistar-Hannover rats. The strain differences in tumor promotion appeared to correlate with inflammatory reactions to quartz and increase of IL-6.

Toxicity of nicotine by repeated intratracheal instillation to f344 rats.

In vivo, nicotine in cigarette smoke induces various effects not only on the respiratory system but also the central and peripheral nerve systems, circulatory organs and digestive organs, and there is a possibility of promotion of lung tumorigenesis. The present experiment was conducted to examine histopathological changes caused by nicotine in the lung with repeated intratracheal instillation (i.t.). Six-week-old male F344 rats were administered nicotine by i.t. at doses of 0.05, 0.1 and 0.2 mg nicotine/rat every 3 weeks beginning at week 4, for up to a total of 9 times and were then sacrificed at week 30. The total number of administrations, total dose of nicotine and effective number of rats were 9 times, 0.45 mg and 5 rats and 4 times, 0.20 mg and 5 rats for the 0.05 mg nicotine/rat group; 3 times, 0.30 mg and 5 rats and 4 times, 0.40 mg and 3 rats for the 0.1 mg group; and 3 times, 0.60 mg and 3 rats for the 0.2 mg group, respectively. As a control group, 5 rats were administered 0.2 ml saline/rat 9 times. Some rats administered 0.1 and 0.2 mg nicotine suffered convulsions just after administration. Histopathologically, though proliferative changes were not observed, neutrophil infiltration, edema and fibrosis in the lung were induced by nicotine. In conclusion, repeated treatment of nicotine promoted neurologic symptoms in the acute phase, and strong inflammation in the lungs in the chronic phase, even at a low dose. Toxicity of nicotine is suggested to depend not on total dose of nicotine in the experiment but rather on repeated injury with consecutive administration.

Lack of promoting effects from physical pulmonary collapse in a female A/J mouse lung tumor initiated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with remarkable mesothelial cell reactions in the thoracic cavity by the polymer.

Experimental identification of potential chemopreventive or tumor promotive agents in the lung is important. Establishment of short-term bioassay models is therefore a high priority. In an attempt to induce strong promotion effects, in Experiment 1, left thoracotomy was performed on A/J mice at week 3 after initiation with 4-(methylnitrosamno)-1-(3-pyridyl)-1-butanone (NNK) (2mg/0.1 ml saline/mouse i.p.) at weeks 0 and 1. In Experiment 2, at week 3, 0.2 ml of polymer gel was infused directly into the left cavity of the thorax with thoracotomy to occupy certain thoracic cavity volume and to examine the influence of physical pulmonary collapse. The experiments were terminated after 8, 10, 12 and 16 weeks in Experiment 1, and 12 weeks in Experiment 2 but no clear promotion effects in either experiment or pulmonary collapse due to infused polymer were apparent. However, a pronounced mesothelial cell reaction to the infused polymer was evident on the left lung surfaces and parietal pleura in Experiment 2. In conclusion, the present experiments did not demonstrate any clear lung tumor promotion effects of thoracotomy or physical left lung collapse. It remains possible, however, that alternative approaches might have greater efficacy and these need more consideration. In addition, mesothelial cells reaction was observed with the infused polymer.

Risk analysis of environmental chemicals on lung carcinogenesis.

Lung cancer is one of the most common cancers in the world, and the incidence of lung cancer is increasing. Risk analysis of environmental chemicals on lung carcinogenesis is particularly important. Detection of chemopreventive agents of lung carcinogenesis is also important to reduce our risk of lung cancer. For that purpose, it is necessary to establish reliable in vivo animal models of lung carcinogenesis. The A/J mouse is a mouse strain sensitive to lung carcinogens, and also develops spontaneous lung tumors without any chemical treatment. We have demonstrated that a treatment of 4-(methylnitrosamino)-1-(3-pyridyle)-1-butanone (NNK), a tobacco specific nitrosamine, or 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (MeIQx), a heterocyclic amine, induced lung tumors in the female A/J mouse in 16 and 32 weeks. The lung tumors developed in the A/J mouse are histopathologically classified as adenocarcinomas, adenomas, and alveolar cell hyperplasias. Some of these types of lung cancer are similar to those of human lung cancer. We also investigated the chemopreventive effects of bovine LF (bLF) on different phases of NNK-induced lung tumorigenesis in A/J mice. The A/J mouse is very useful mouse strain as a reliable in vivo model, which can be used for risk analysis of lung carcinogenesis.