Safety and efficacy of PD-1/PD-L1 blockade in patients with preexisting antinuclear antibodies.

PURPOSE: Immune checkpoint inhibitors (ICIs) show promising clinical activity in advanced cancers. However, the safety and efficacy of PD-1/PD-L1 blockade in patients with preexisting antinuclear antibodies (ANA) are unclear. METHODS: 191 patients treated with nivolumab, pembrolizumab, atezolizumab, or durvalumab for unresectable advanced cancers between September 2014 and December 2018 were identified retrospectively. Patients were divided into positive (ANA titers ≥ 1:160) and negative ANA groups (ANA titers < 1:160). Development of immune-related adverse events (irAEs), the overall response rate (ORR), and disease control rate (DCR) were monitored. RESULTS: Positive ANA titers were seen in 9 out of 191 patients. Four patients in the positive ANA group and 69 patients in the negative group developed irAEs of any grade without a significant difference between the groups. The development of endocrine, pulmonary, and cutaneous irAEs was not significant, whereas positive ANA was significantly higher in patients who developed colitis (2/9) than in patients who did not (3/182, P = 0.0002). DCR in the positive and negative ANA group was 37.5% and 67.5%, respectively, and was not statistically significant, but had better efficacy in patients without ANA (P = 0.08). ANA-related autoimmune diseases such as SLE, Sjögren's syndrome, MCTD, scleroderma, dermatomyositis, and polymyositis was not induced in either group. However, one patient with preexisting dermatomyositis had a flare up after initiation of atezolizumab. CONCLUSION: Further studies to identify predictive factors for the development of irAEs are required to provide relevant patient care and maximize the therapeutic benefits of ICIs.

Spontaneous hepatic copper accumulation in Long-Evans Cinnamon rats with hereditary hepatitis. A model of Wilson's disease.

Long-Evans Cinnamon (LEC) rats, an inbred strain of a mutant rat isolated from Long-Evans rats, develop hereditary hepatitis. To elucidate the role of copper metabolism in the development of the hepatitis in LEC rats, we examined the copper concentration in the tissues and serum levels of copper and ceruloplasmin. Copper concentration in the liver of LEC rats was over 40 times that of normal Long-Evans Agouti (LEA) rats, while the serum ceruloplasmin and copper concentrations in LEC rats decreased significantly. The hepatocytes of LEC rats show steatosis in cytoplasm and pleomorphism of mitochondria, resembling the histologic features of the liver in Wilson's disease. These findings suggest that the hereditary hepatitis in LEC rats is closely associated with copper toxicity, and may be dealing with a rat form of Wilson's disease. Thus the LEC rats will provide a unique and useful animal model for clarifying the mechanism and for developing treatment strategies for Wilson's disease and other abnormal copper metabolism in humans.

Vascular endothelial growth factor promoter-based conditionally replicative adenoviruses for pan-carcinoma application.

Treatment of advanced lung cancer is one of the major challenges in current medicine because of the high morbidity and mortality of the disease. Advanced stage lung cancer is refractory to conventional therapies and has an extremely poor prognosis. Thus, new therapeutic approaches are needed. Lung tumor formation depends on angiogenesis in which the vascular endothelial growth factor (VEGF) produced by cancer cells plays a pivotal role. Neutralizing VEGF with a soluble VEGF receptor suppresses tumor growth; however, the anticancer effect with this therapy is weakened after the intratumoral vascular network is completed. In this study, we turned the expression of VEGF by tumors to therapeutic advantage using a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 is controlled by the human VEGF promoter. This virus achieved good levels of viral replication in lung cancer cells and induced a substantial anticancer effect in vitro and in vivo. As a further enhancement, the cancer cell killing effect was improved with tropism modification of the virus to express the knob domain of Ad3, which improved infectivity for cancer cells. These VEGF promoter-based CRAds also showed a significant cell killing effect for various types of cancer lines other than lung cancer. Conversely, the VEGF promoter has low activity in normal tissues, and the CRAd caused no damage to normal bronchial epithelial cells. Since tumor-associated angiogenesis via VEGF signalling is common in many types of cancers, these CRAds may be applicable to a wide range of tumors. We concluded that VEGF promoter-based CRAds have the potential to be an effective strategy for cancer treatment.

Prevention of radiation-induced pneumonitis by recombinant adenovirus-mediated transferring of soluble TGF-beta type II receptor gene.

To investigate whether radiation-induced pneumonitis in the mouse-irradiated lung could be prevented by recombinant adenovirus-mediated soluble transforming growth factor-beta (TGF-beta) type II receptor gene therapy. Radiation fibrosis-prone mice (C57BL/6J) were randomly divided into four groups consisting of a (1) control group (sham-irradiated); (2) radiation (RT)-alone group; (3) RT+AdCMVsTbetaR group and (4) RT+AdCMVluc group. The RT-alone and sham-irradiated mice were killed at several time points after thoracic irradiation with a single dose of 9 Gy, and then the TGF-beta1 concentrations in serum and broncho-alveolar lavage fluid (BALF) were quantified by enzyme-linked immunosorbent assay (ELISA). We used an adenoviral vector expressing a soluble TGF-beta type II receptor (AdCMVsTbetaR), which can bind to TGF-beta and then block the TGF-beta receptor-mediated signal transduction. The C57BL/6J mice were intraperitoneally (i.p.) injected with either 5 x 10(8) plaque-forming units of AdCMVsTbetaR or AdCMVluc, a control adenovirus-expressing luciferase, a week preceding and a week following the X-ray thoracic irradiation. Four weeks after irradiation, the mice were killed and the concentration of TGF-beta1 in the serum and BALF were then measured using ELISA and the lung tissue specimens were examined histopathologically. Following thoracic irradiation with a single dose of 9 Gy, radiation-induced TGF-beta1 release in the serum reached the first peak concentration at 12 h and then declined. It reached a maximal value at 2 weeks after irradiation. In the BALF, the TGF-beta1 concentration was appreciable within the first hour and thereafter declined. It reached a maximal value at 3 days after irradiation. A one-time i.p. injection of AdCMVsTbetaR 1 week before irradiation could not completely suppress the two peaks of the radiation-induced TGF-beta1 increase, whereas an injection a week preceding and a week following thoracic irradiation was able to suppress those two peaks thoroughly. The TGF-beta1 was completely suppressed in the AdCMVsTbetaR-treated mouse serum and BALF; however, no statistical difference was observed in the serum and BALF between the AdCMVluc-infected mice and the control mice at 4 weeks after irradiation (P < 0.05). A histopathological examination showed only mild radiation pneumonitis in the irradiated lungs of AdCMVsTbetaR-treated mice in comparison to the AdCMVluc-infected and RT-alone mice. Our results demonstrated that TGF-beta1 plays an important role in radiation pneumonitis, thus suggesting that the adenovirus-mediated overexpression in soluble TGF-beta type II receptor gene therapy may be a potentially feasible and effective strategy for the prevention of radiation pneumonitis.

Genomic organization of human fetal specific P-450IIIA7 (cytochrome P-450HFLa)-related gene(s) and interaction of transcriptional regulatory factor with its DNA element in the 5' flanking region.

P-450IIIA7 is a form of cytochrome P-450 which was isolated from human fetal livers and termed P-450HFLa. This form has been clarified to be expressed during fetal life specifically (Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. and Kamataki, T. (1990) Biochemistry 29, 4430-4433). In the present study, we isolated five independent clones which probably corresponded to the human P-450IIIA7 gene. These clones were completely sequenced, all exons, exon-intron junctions and the 5' flanking region from the cap site to-869. Although the sequences in the coding region were completely identical to P-450IIIA7, it is possible that genomic fragments sequenced in this study encode portions of other P-450IIIA7-related genes since we could not obtain a complete overlapping set of genomic clones. Within its 5' flanking sequence, the putative binding sites of several transcriptional regulatory factors existed. Among them, it was shown that a basic transcription element binding factor (BTEB) actually interacted with the 5' flanking region of this gene.

Detection of tyrosine hydroxylase mRNA and minimal neuroblastoma cells by the reverse transcription-polymerase chain reaction.

To facilitate the diagnosis of bone marrow metastasis in neuroblastoma, we have developed a method of amplifying and detecting the tyrosine hydroxylase (TH) mRNA sequence in bone marrow cells using a combination of reverse transcription and the polymerase chain reaction (RT/PCR). By this method, the sequence of TH was detected clearly in the neuroblastoma tissues of all 6 patients and not detected in the bone marrow cells of any of the 9 negative control children. In a reconstitution experiment, 1 neuroblastoma cell per 100,000 normal bone marrow cells could be detected, thus indicating the great sensitivity of this method. Based on these results, this technique may be of value in the diagnosis and treatment follow-up of bone marrow metastasis of neuroblastoma.

Comparison of chemotherapy with or without medroxyprogesterone acetate for advanced or recurrent breast cancer.

The usefulness of CAF [cyclophosphamide (CPA)/doxorubicin (ADR)/5-fluorouracil (5-FU)] + medroxyprogesterone acetate (MPA) therapy for advanced/recurrent breast cancer was studied in a randomised trial at 56 institutions. Patients received CAF therapy [CPA: 100 mg, orally, days 1-14; ADR: 30 mg/m2, intravenously (i.v.), days 1 and 8; 5-FU: 500 mg/m2, i.v., days 1 and 8) in arm I, or CAF + MPA therapy (CAF + MPA 1200 mg, daily) in arm II. The response rate was significantly higher (P = 0.041) in arm II (53.5%, 46/86) than arm I (36.6%, 30/82). The response rate by tumour site was significantly higher for lymph node and bone lesions in arm II. Partial response duration and overall response duration were significantly longer in arm II. Incidences of anorexia and nausea/vomiting were significantly higher in arm I but in arm II, moon face, oedema and vaginal bleeding were significantly higher. Many patients in arm II demonstrated improvement in performance status and weight loss, suggesting a beneficial effect of MPA. The chemoendocrine therapy with CAF + MPA appears to be more beneficial than CAF alone in the treatment of advanced/recurrent breast cancer.

Increased expression of c-jun gene during spontaneous hepatocarcinogenesis in LEC rats.

We have studied the expressions of nine proto-oncogenes (c-myc, N-myc, c-fos, C-jun, p53, H-ras, N-ras, c-raf, hst) and two other genes (PCNA, GST-P) during the spontaneous development of hepatocellular carcinomas (HCCs) in LEC rats. Expression of c-myc, H-ras, N-ras, C-raf, p53 and PCNA genes was detected, but this did not significantly change during the development of HCCs in LEC rats. Expression of N-myc and hst genes was not detectable. Expression of c-fos gene was detected in one HCC case out of four. Significantly increased expression of c-jun gene was observed in the liver tissues of LEC rats aged 8 months. This high expression was decreased with the development of HCCs. On the other hand, the expression of GST-P gene increased in parallel with the clinical course of the development of HCCs in LEC rats. The pattern of c-jun mRNA augmentation was different from that of GST-P mRNA. These observations suggest that c-jun gene may play a role in the spontaneous development of HCCs in LEC rats.

Serum levels of the type IV collagen 7s domain in patients with chronic viral liver diseases.

The serum levels of type IV collagen 7s domain (7s collagen) were determined in 80 patients with hepatocellular carcinoma (HCC) and in 105 with chronic liver disease without HCC. Among 86 patients with HCV infection, serum levels of the 7s collagen were significantly higher in those with HCC than-in those without HCC (p<0.05). In contrast, no significant differences were recognized between these two groups in 84 patients with HBV infection. Patients with HCV infection having high serum levels of the 7s collagen exceeding 7.0 ng/ml, were diagnosed as HCC with a sensitivity of 71.8%, a specificity of 74.5%, and a reliability of 77.9%. It was concluded that the progression of hepatic fibrosis plays an important role in the hepatocarcinogenesis of HCV, and that serum levels of the 7s collagen appeared to be useful as a risk factor of the development of HCV-related HCC.

Postoperative immunocompetence and immunosuppressive effect of rat liver tumors.

To investigate the kinetics of postoperative immunocompetence and immunosuppressive effects of rat liver tumors, we examined natural killer cell cytotoxicity and blastogeneses to phytohemagllutinin-P, concanavalin A, and lipopolysaccharide on 1-, 2-, 3-, 5-, 7-, 9- and 14- postoperative days (-POD) after partial hepatectomy in tumor-bearing or healthy rats. The immunosuppressive effects of the tumor were observed on all the parameters examined, particularly from 1- to 3-POD and on 14-POD. Surgical stress induced immunosuppression in the early postoperative stage which lasted over 1 week, and the immunosuppression was seriously affected by the presence of the tumor. The present study suggests that surgery without curability is closely related to enhancement of the growth of the remaining or occult cancer, leading to worse survival.

Hepatocyte growth factor enhances the invasion activity of human hepatocellular carcinoma cell lines.

We investigated whether hepatocyte growth factor (HGF) enhances the invasion activity of three human HCC cell lines, HLF, HLE, and HC-4, in vitro. The analysis of the invasiveness consisted of the production of u-PA and the chemotaxis for fibronectin. Invasion activity of all cell lines was enhanced by the addition of recombinant human hepatocyte growth factor (rhHGF) to the medium. HGF stimulated the production of u-PA in HLF cells. HGF accelerated the chemotaxis of HC-4 and HLE. These data suggest that HGF increase the invasion activity of human HCC cell lines by affecting the production of u-PA or the chemotaxis for fibronectin.

Expression of sex-hormone receptors and clinicopathological findings in hepatocellular-carcinoma.

We studied the expresssion of estrogen (ER), progesterone (PgR), and androgen receptors (AR) in hepatocellular carcinoma (HCC) and found expression in (7/36), 3.2% (1/31), 16.7% (4/24) of cases, respectively. The expression of ER did not correlate with histological grade, tumor size, or stage of disease. On the other hand, all the patients with AR-positive tumors had stage IV disease, and only 25% of the patients with AR-negative tumors had stage IV disease. The survival was not influenced by the expression pattern of ER, however, the survival of the patients with AR-positive tumors tended to be worse than that of patients with AR-negative tumors. We suggest that the AR expression correlates better with poor outcome in HCC than the ER expression.

Co-cultured endothelial and Kupffer cells regulate hepatocyte replication.

We studied the effect of co-culture with endothelial and/or Kupffer cells on hepatocyte replication in primary culture by means of [H-3]-thymidine incorporation. Hepatocytes, sinusoidal endothelial and Kupffer cells were extracted from rat liver by a two-step perfusion and subsequent two-step elutriation method. The replication was promoted in proportion with the densities of endothelial cells, but inversely suppressed by co-culture with Kupffer cells. The replicative effect of endothelial cells was not observed with the high number of hepatocytes cultured, however. The hepatocyte replication was also regulated in accordance with the ratios of endothelial/Kupffer cells. These results indicate that endothelial cells have a positive effect and Kupffer cells have a negative effect on hepatocyte replication, and that this replication may be associated with hepatocarcinogenesis and/or tumor-growth of hepatocellular carcinoma.

Surgical procedures in gallbladder cancer with special reference to its macroscopic appearance.

To investigate the relationships of the gross appearance to cancer depth and microinvasion in gallbladder cancer, thirty-three cancers were macroscopically subgrouped: papillary (n=8), nodular (n=6), papillo-invasive (n=1), nodulo-invasive (n=11), and invasive (n=7) type, respectively. Papillary type cancers were limited to the mucosa or the muscularis propria, without microinvasion. Most nodular type cancers infiltrated to the subserosa with moderate microinvasion. The other types of cancer frequently invaded the subserosa or more, with high incidence of microinvasion. Only the papillary type cancers indicated simple cholecystectomy, the other types should be operated in accordance with infiltration depth and degree of involvement.

Fluted serum relating to growth of both hepatocyte and hepatocellular carcinoma cells.

This study was undertaken to investigate a humoral substance contributing to both primary hepatocytes and hepatocellular carcinoma (HCC) cells in the primitive form. Peripheral blood from rat, 24 h after partial hepatectomy (PH) or sham-operation, was eluted into 50 fractions using an HPLC, and each fraction was bio-assayed. In vivo relationship between PH and tumor growth was also evaluated. Only one, approximately 100 kDa, of the 50 fractions from PH rat stimulated the growth of both types of cell, and the time-lag between PH and HCC injection also correlated significantly with tumor appearance and growth. Serum in PH rats contains certain humoral stimulator(s) contributing to growth of both hepatocyte and HCC cells.

Prediction of prognosis after resection in human hepatomas by nuclear-DNA analysis.

Nuclear DNA analysis with flow cytometry was performed in 81 patients with hepatocellular carcinoma. Thirty-eight cases (46.9%) were diploid type and forty-three cases (53.1%) aneuploid type. Microscopical portal vein invasion of the tumor was more frequent in the aneuploid group (p<0.05). Significantly higher frequency of intrahepatic metastasis was found in the aneuploid group (p<0.05). There was no correlation between the tumor nucleus DNA ploidy pattern and the recurrence rate, 23 (60.5%) of diploid cases and 28 (65.1%) of aneuploid cases, respectively. Disseminated recurrence was more frequent in the aneuploid group than in the diploid group (p<0.10). Significantly better survival rates were observed in the diploid group which had microscopical portal invasion. Moreover, the diploid group which had no capsular formation and intrahepatic metastasis showed significantly longer survival terms.

Accelerated rejection of allografted rat liver perfused with anti-ICAM-1 monoclonal antibody.

Infusion of mouse anti-rat ICAM-1 into the portal vein of ACI rat liver graft before transplantation resulted in accelerated rejection by LEW recipients (2.9 +/- 2.5 days), when compared to the ACI liver grafts perfused with either Lactate-Ringer's solution (9.8 +/- 1.3) or anti-human ICAM-1 mAb (9.8 +/- 3.1). Histologic features of anti-rat ICAM-1 perfused grafts were spotty ischemic necrosis, whereas those of control grafts were marked cellular infiltration. These combined survival and histologic data suggest that the accelerated rejection may be mediated by vascular deterioration.

Pathological comparison between spontaneously developed and chemically induced liver cancers in LEC rats with hereditary hepatitis.

To investigate the pathological differences between spontaneously developed and chemically induced liver cancers in LEC rats with hereditary hepatitis, eight-week-old LEC rats were fed 0.06% or 0.03% of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) for 12 weeks, and then fed basal diet. Spontaneous liver cancers occurred in 60.4% of the LEC rats, while 3'-Me-DAB induced cancers in all LEC rats. The survival periods of chemically (0.06%) treated LEC rats were significantly shorter than non-treated rats. The chemically-induced cancers were strongly related to Edmondson classification. Metastasis and transplantability of the chemically-induced cancers were higher than spontaneous cancers. LEC rats are highly susceptible to the chemical carcinogen with the initiated status of hepatocarcinogenesis.

HLA class I antigens are possible prognostic factors in hepatocellular carcinoma.

Thirty patients who underwent hepatectomy for the treatment of hepatocellular carcinoma (HCC) were examined for expression of HLA class I antigens on HCC cells by flow cytometry. The expression was found significantly lower in cases of stage IV compared with those of stage I or stage II (p<0.05), and in cases of intrahepatic metastases compared with those without metastases (p<0.001). In cases of non-curative hepatectomy, the expression of HLA class I antigens was lower compared with those treated by curative resection. Postoperative cumulative disease-free survival rates were well correlated with the expression rate of HLA class I antigens (p<0.05). Expression of HLA class I antigens on HCC may indicate low malignancy and better prognosis.

Pyrimidine nucleoside phosphorylase-activity and biological characteristics of breast-cancer.

We investigated whether Pyrimidine nucleoside phosphorylase (PyNPase) activity in breast cancer tissue correlated with biological characteristics of breast cancer. PyNPase activity, ER, PgR, EGFR, DNA ploidy pattern, PCNA positive cells and amplification of the c-erbB-2 gene were determined in specimens from 138 patients. PyNPase activity was significantly higher in ER negative than ER positive carcinomas (p<0.05), in PgR negative than PgR positive carcinomas (p<0.05) and significantly higher in tumors with c-erbB-2 gene amplification compared with tumors with no amplification (p<0.05). The results suggest that PyNPase activity in breast cancer tissue may be a new biological characteristic of breast cancer.