Tumor immune microenvironment and nivolumab efficacy in EGFR mutation-positive non-small-cell lung cancer based on T790M status after disease progression during EGFR-TKI treatment.

BACKGROUND: The efficacy of programmed death-1 blockade in epidermal growth factor receptor gene (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) patients with different mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) is unknown. We retrospectively evaluated nivolumab efficacy and immune-related factors in such patients according to their status for the T790M resistance mutation of EGFR. PATIENTS AND METHODS: We identified 25 patients with EGFR mutation-positive NSCLC who were treated with nivolumab after disease progression during EGFR-TKI treatment (cohort A). Programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) density in tumor specimens obtained after acquisition of EGFR-TKI resistance were determined by immunohistochemistry. Whole-exome sequencing of tumor DNA was carried out to identify gene alterations. The relation of T790M status to PD-L1 expression or TIL density was also examined in an independent cohort of 60 patients (cohort B). RESULTS: In cohort A, median progression-free survival (PFS) was 2.1 and 1.3 months for T790M-negative and T790M-positive patients, respectively (P = 0.099; hazard ratio of 0.48 with a 95% confidence interval of 0.20-1.24). Median PFS was 2.1 and 1.3 months for patients with a PD-L1 expression level of ≥1% or <1%, respectively (P = 0.084; hazard ratio of 0.37, 95% confidence interval of 0.10-1.21). PFS tended to increase as the PD-L1 expression level increased with cutoff values of ≥10% and ≥50%. The proportion of tumors with a PD-L1 level of ≥10% or ≥50% was higher among T790M-negative patients than among T790M-positive patients of both cohorts A and B. Nivolumab responders had a significantly higher CD8+ TIL density and nonsynonymous mutation burden. CONCLUSION: T790M-negative patients with EGFR mutation-positive NSCLC are more likely to benefit from nivolumab after EGFR-TKI treatment, possibly as a result of a higher PD-L1 expression level, than are T790M-positive patients.

Clinical application of amplicon-based next-generation sequencing to therapeutic decision making in lung cancer.

BACKGROUND: The clinical implementation of genomic profiling for lung cancer with high-throughput, multiplex tests is warranted to allow prioritization of appropriate therapies for individual patients. We have now applied such testing to detect actionable mutations that may inform treatment recommendations in lung cancer. PATIENTS AND METHODS: We prospectively applied amplicon sequencing panels that cover both mutational hotspots in 22 genes related to lung and colon tumorigenesis as well as 72 major variants of ALK, RET, ROS1, and NTRK1 fusion transcripts. We then determined the proportion of patients who received genotype-directed therapy and their overall survival (OS). RESULTS: Tumor specimens from 110 patients with lung cancer recruited between July 2013 and March 2015 were analyzed. The most common genetic alterations were TP53 mutations in 42 patients, followed by EGFR mutations in 25, STK11 mutations in 12, and KRAS mutations in 10. Potentially actionable mutations were identified in 44 patients including 50% of those with adenocarcinoma and 14% of those with squamous cell carcinoma. The OS of patients with advanced or recurrent cancer who had an actionable mutation and received targeted therapy (median OS not achieved) was significantly longer than that of those with no mutation (18.1 months, P = 0.041) or of those with a mutation not so treated (6.1 months, P = 0.0027). CONCLUSIONS: Multiplex genomic testing was performed on formalin-fixed, paraffin-embedded tumor specimens with a success rate of ≥95%. Such testing can assist physicians in matching patients with approved or experimental targeted treatments. CLINICAL TRIAL REGISTRATION: The University Medical Hospital Information Network (UMIN) Clinical Trials Registry under the identifier UMIN000014782.

Molecular analysis of two enzyme genes, HPRT1 and PRPS1, causing X-linked inborn errors of purine metabolism.

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. On the other hand, PRPS1 mutations cause PRPP synthetase superactivity associated with hyperuricemia and gout, sometimes including neurodevelopmental abnormalities. We have identified two mutations in two Lesch-Nyhan families after our last report. One of them, a new single nucleotide substitution (130G>T) resulting in a missense mutation D44Y was detected in exon 2 of HPRT1. RT-PCR amplification showed not only a cDNA fragment with normal size, but also a small amount of shorter fragment skipping exons 2 and 3. The other missense mutation F74L (222C > A) was detected in a Japanese patient but has been reported previously in European families. In four hyperuricemic patients with mild neurological abnormality, no mutations responsible for partial HPRT deficiency were identified in HPRT1. In these four patients, we also performed molecular analysis of PRPS1, but no mutations in PRPP synthetase were found.

Identification of a metastasis signature and the DLX4 homeobox protein as a regulator of metastasis by combined transcriptome approach.

Although widespread metastasis is the major cause of human lung cancer-related deaths, its underlying mechanism remains largely unclear. Our genome-wide comparison of the expression profiles of a highly metastatic lung cancer cell line, NCI-H460-LNM35 (LNM35), and its parental clone, NCI-H460-N15 (N15), resulted in the identification of a cancer metastasis signature composed of 45 genes. Through gene ontology analysis, our study also provided insights into how this 45-gene metastasis signature may contribute to the acquisition of metastatic potential. By applying the signature to datasets of human cancer cases, we could demonstrate significant associations with a subset of cases with poor prognosis not only for the two datasets of cancers of the lung but also for cancers of the breast. Furthermore, we were able to show that enforced expression of the DLX4 homeobox gene, which was identified as a gene with significant downregulation in LNM35 as well as with significant association with favorable prognosis for lung cancer patients, markedly inhibited in vitro motility and invasion as well as in vivo metastasis via both hematogenous and lymphogenous routes. Taken together, these findings indicate that our combined transcriptome analysis is an efficient approach in the search for genes possessing both clinical usefulness in terms of prognostic prediction in human cancer cases and clear functional relevance for studying cancer biology in relation to metastasis.

Long-term ursodeoxycholic acid therapy is associated with reduced risk of biliary pain and acute cholecystitis in patients with gallbladder stones: a cohort analysis.

Whether ursodeoxycholic acid (UDCA) therapy alters the long-term clinical course of gallstones (GS) without stone dissolution remains unknown. We aimed to clarify the relationship between long-term UDCA therapy and risks of biliary pain or acute cholecystitis in GS patients. We also aimed to identify factors affecting the natural course, and to explore a simple patient selection criteria for UDCA therapy. A cohort of 527 uncomplicated GS patients with or without UDCA (600 mg/d) followed for up to 18 years was analyzed. Patients who had frequent attacks or were complicated with cholecystitis were converted to cholecystectomy. History and UDCA therapy were identified on Cox analysis as 2 factors affecting the long-term clinical course. In patients without therapy, history was the only predictor of biliary pain among various patient or stone characteristics; biliary pain was rare in asymptomatic patients, while frequent in symptomatic patients (P <.001). UDCA therapy was associated with reduced risk for biliary pain in both symptomatic (62% vs. 92% in untreated patients at 10 years; P <.001; relative risk, 0.19; 95% CI, 0.10-0.34) and asymptomatic patients (6% vs. 12% in untreated patients at 10 years; P =.037; relative risk, 0.19; 95% CI, 0.04-0.91). Risk for the conversion was also reduced in UDCA-treated symptomatic patients (26% vs. 88% in untreated patients at 10 years, P <.001; relative risk, 0.08; 95% CI, 0.03-0.22). These effects were independent of stone dissolution. Three factors were identified on Cox analysis as affecting GS dissolution: radiolucency, small size (<10 mm) of stones, and visualized gallbladder (GB) on cholecystogram. A selection criteria based on these appears to exhibit high sensitivity (74%) and specificity (95%) for dissolution. UDCA therapy might be considered in symptomatic patients fulfilling these criteria, and also in patients who have significant surgical risk, because the longterm therapy is clearly associated with reduced risk of biliary pain and acute cholecystitis.

Construction of COD simulation model for activated sludge process by fuzzy neural network.

Fuzzy neural network (FNN) was applied to construct a simulation model for estimating the effluent chemical oxygen demand (COD) value of an activated sludge process in a "U" plant, in which most of process variables were measured once an hour. The constructed FNN model could simulate periodic changes in COD with high accuracy. Comparing the simulation result obtained using the FNN model with that obtained using the multiple regression analysis (MRA) model, it was found that the FNN model had 3.7 times higher accuracy than the MRA model. The FNN models corresponding to each of the four seasons were also constructed. Analyzing the fuzzy rules acquired from the FNN models after learning, the operational characteristic of this plant could be elucidated. Construction of the simulation model for another plant "A", in which process variables were measured once a day, was also carried out. This FNN model also had a relatively high accuracy.

Oral injury and mouthguard usage by athletes in Japan.

The use of mouthguards in contact sports effectively prevents oral injury and preserves oral structures. We investigated oral injuries and awareness concerning mouthguards among Japanese high school soccer and rugby players. Athletes were asked a series of questions concerning their history of oral injury while participating in sports, and their pattern of mouthguard use was determined. The data were evaluated statistically using chi-square analysis. The incidence of oral injuries was 32.3% for soccer athletes and 56.5% for rugby athletes, with 0.8% and 24.1% of the respective groups having mouthguards. There were significant differences between the soccer and rugby groups (P < 0.001). Although 81.8% of soccer athletes thought mouthguards were unnecessary, only 26.2% of rugby athletes shared this opinion and there was a significant difference between the soccer and rugby groups. Many soccer athletes had insufficient knowledge about mouthguards and were not concerned about preventing oral injury, although it was in fact a common problem in their sport. Athletes as well as coaches must be made aware of the high risk of oral injury when playing soccer, rugby, and other contact sports.

Severe acute hepatitis A associated with acute pure red cell aplasia.

A rare case of severe acute hepatitis A complicated by pure red cell aplasia (PRCA) is reported. A 60-year-old man with jaundice and hepatomegaly was diagnosed as having acute hepatitis A by positive IgM anti-hepatitis A antibody (anti-HAV). Severe anemia rapidly developed 3 weeks after admission, and the patient was diagnosed with PRCA by both bone marrow smears and erythrocyte survival study. The anemia was transient and bone marrow recovered within 1 week. However, concomitant with bone marrow recovery, the hepatitis worsened. He became drowsy and disoriented and severe jaundice, ascites, prolonged prothrombin time, increased transaminase levels, and abnormal electroencephalogram (EEG) were exhibited. Plasma exchange transfusion and glucagon-insulin (GI) therapy improved the consciousness level, but bilirubin, transaminase levels, and IgM anti-HAV titer remained high. Intravenous administration of lipophilized prostaglandin E1 (lipo-PGE1) was added to the GI therapy. Bilirubin and transaminase levels were normalized in the 8th week after the initiation of this combination therapy (17 weeks after admission). The combined use of lipo-PGE1 with plasma exchange and GI therapy appeared to be useful for the prolonged severe hepatitis in this patient.

Immunosuppression by anti-ICAM-1 and anti-LFA-1 monoclonal antibodies of free and vascularized skin allograft rejection.

Immunosuppression by anti-adhesion molecule antibody of free or vascularized skin allograft rejection was investigated in rats. Lewis (LEW, RT11) rats were used as donors and Fisher (F344, RT11v1) rats as the recipients. When F344 rats were treated intraperitoneally (i.p.) with anti-intercellular adhesion molecule-1 (ICAM-1) mAb (1A29) (3 mg/kg/day) and anti-leukocyte function-associated antigen-1 (LFA-1) mAb (WT.1) (3 mg/kg/day) one day prior to grafting and daily after grafting for nine days, free skin graft survival was prolonged only slightly compared with that in control rats which were injected i.p. with a daily dose of 6 mg/kg of anti-TNP mAbs (H1-6-2) one day prior to grafting and daily after grafting for nine days. (Mean survival time [MST] of the free skin graft was 11.2 +/- 0.6 days in the control group and 13.4 +/- 0.3 days in the 1A29 + WT-1 treated group [p < 0.01], respectively.) On the other hand, the vascularized graft survival was prolonged significantly in anti-ICAM-1/LFA-1 mAbs-treated F344 rats as compared with that in control rats. (The mean vascularized graft survival time was 14.2 +/- 0.7 days in the control group and 21.5 +/- 1.9 days in 1A29 + WT-1 treated group [p < 0.002]). Our results suggest that interaction with ICAM-1 and LFA-1 is more important in the rejection of vascularized skin allografts than that of free skin allografts.

Evidence for the early recruitment of T-cell receptor gamma delta+ T cells during rat listeriosis.

We have previously reported that heat-shock protein (hsp) 60-reactive T-cell receptor (TCR)gamma delta+ T cells appear in the peritoneal cavity during the early stage of infection with Listeria monocytogenes in mice. In this study, we examined the kinetics of TCR gamma delta+ T cells during listeriosis in F344 rats by flow cytometry using a V65 monoclonal antibody (mAb) directed to a constant determinant of rat TCR gamma delta chains. TCR gamma delta+ T cells significantly increased in the peritoneal cavity on day 6 and then decreased by day 10 after infection, in parallel with the kinetics of hsp60 expression in the peritoneal macrophages during listeriosis in F344 rats. Most of the early appearing TCR gamma delta+ T cells were of the CD4- CD8 alpha beta+ CD5+ lymphocyte function-associated antigen (LFA)-1 alpha high CD45RC- interleukin-2 receptor (IL-2R) alpha- phenotype, although a significant fraction of the TCR gamma delta+ T cells expressed CD8 alpha only. The increase in TCR gamma delta+ T cells during listeriosis was prominent in F1 (F344 x Lewis) rats but only marginal in Lewis rats, which was correlated with the expression level of hsp 60 in the peritoneal macrophages. The peritoneal TCR gamma delta+ T cells in naive F344 rats appeared to proliferate significantly in response to recombinant hsp 60 (rhsp 60) derived from Mycobacterium bovis bacillus Calmette-Guérin (BCG). These results imply that the early appearance of hsp 60-reactive TCR gamma delta+ T cells during listerial infection can be generalized across species.

Antibodies against leukocyte function-associated antigen-1 and against intercellular adhesion molecule-1 together suppress the progression of experimental allergic encephalomyelitis.

We obtained the evidence that coadministration in vivo of mAbs against leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) suppressed the progression of experimental allergic encephalomyelitis (EAE) in rats. The suppressive effect in vivo of coadministration of the mAbs during the induction phase was not prominent, but the administration of these mAbs during the effector phase markedly suppressed the progression of clinical illness and prevented the infiltration of encephalitogenic cells into the central nervous system. However, administration of the mAb to LFA-1 alone or ICAM-1 alone did not show such suppressive effects. These findings suggest that LFA-1 and ICAM-1 are critically involved in the development of EAE and that the administration together of mAbs against adhesion molecules including LFA-1 and ICAM-1 might provide a new immunotherapeutic approach for the treatment of multiple sclerosis.

Intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 are involved in protection mediated by CD3+TCR alpha beta- T cells at the early stage after infection with Listeria monocytogenes in rats.

To investigate the significance of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1 (LFA-1) in host defense against infection with intracellular parasites, we examined the effects of in vivo pretreatment with mAbs to ICAM-1 (1A29) and LFA-1 alpha (WT-1) on the protection against infection with Listeria monocytogenes in Fisher F344/N rats. Expression of ICAM-1 and LFA-1 alpha molecules on T cells in spleen, liver and peritoneal cavity of rats was down-regulated after i.p. administration with daily doses of 300 micrograms of either 1A29 or WT-1 for 10 days. The survival rate of rats inoculated with viable Listeria was significantly reduced by in vivo pretreatment with 1A29 together with WT-1 for 10 days but not by in vivo pretreatment with control mAb. The numbers of bacteria in the spleen in rats pretreated with both 1A29 and WT-1 were significantly increased on day 3 and day 6 after infection with 1 x 10(7) of viable Listeria corresponding to 1/30 of LD50 to normal rats. Thus, the resistance against listerial infection was severely impaired by combinational pretreatment with mAbs in ICAM-1 and LFA-1 alpha. As shown in our previous report, the early appearance of CD3+TCR alpha beta- T cells, presumably TCR gamma delta T cells, was evident in the peritoneal cavity and liver of control rats at the early stage after listerial infection, while this was suppressed at this stage in rats pretreated with both 1A29 and WT1.(ABSTRACT TRUNCATED AT 250 WORDS)

Delay in expression of a mammary tumor provirus is responsible for defective clonal deletion during postnatal period.

A gene-encoding ligand for deletion of T cells bearing TcRV beta 6 and V beta 8.1 cosegregates a new mammary tumor provirus locus, Mtv-50 in NC mice. The sequence of the open reading frame (ORF) in the 3' long terminal repeat (LTR) of Mtv-50 was strikingly similar to those of Mtv-7, Mtv-43 and exogenous mouse mammary tumor virus (SW) with properties of minor lymphocyte stimulating antigen 1a. Consistent with previous reports, clonal deletion of mature thymocytes bearing TcRV beta 6 was defective during the early postnatal period of mice carrying Mtv-50. Appreciable levels of mRNA corresponding to common Mtv ORF and Mtv-6 ORF were expressed in the neonatal thymus, while little, if any, mRNA corresponding to Mtv-50 ORF was detected in the thymus at the early postnatal stage. Delay in expression of Mtv-50 ORF during the postnatal period may be responsible for the failure of clonal deletion of V beta 6-T cells in the early postnatal life of mice carrying Mtv-50.

[A study on reduction rate of tumor volume by irradiation in germinoma, malignant lymphoma, and medulloblastoma].

A relationship between radiation dose and reduction rate of tumor volume was studied in 10 patients with germinoma, nine with malignant lymphoma, and seven with medulloblastoma, in order to evaluate the effect of irradiation on these tumors. Germinomas showed either mono- or biphasic reduction of the volume with increased radiation dose in semi-logarithmic expression, irrespective of their size. All patients are well without relapse except for one that was lost because of transfer. The reduction rate of tumor volume was slightly less in malignant lymphomas and medulloblastomas than that in germinomas and the former two showed less uniformity of response to irradiation according to each case. Malignant lymphomas tended to regrow during irradiation period or early after the period and the effect of irradiation seemed to be related to the size of the tumor. Medulloblastomas did not completely disappeared during irradiation period, but delayed effect of irradiation was recognized after the period.

Cumulative effect of repeated ischemia on brain edema in the gerbil. Biochemical and physiological correlates of repeated ischemic insults.

Using a bilateral carotid artery occlusion model in the gerbil, we evaluated the cumulative effect of repeated ischemic insults on various physiological and biochemical parameters in brain. The most striking consequence of repeated occlusions is a profound, delayed increase in brain edema between 6- and 24-hr recirculation, after a series of three 5-min occlusions carried out at 1-hr intervals. This increment in brain water is accompanied by morphological evidence of compressed capillaries with increased filling of larger vessels, consistent with impaired microcirculation even though total blood flow and total vascular volume return toward control levels. The effect on edema is most severe when occlusions are repeated during the period of postischemic hypoperfusion, although the mechanisms responsible for this effect remain to be determined. Histograms of cerebral cortical oxygen tension show a shift toward progressively lower values during the recirculation interval after each occlusion, suggesting that secondary hypoxia accompanying hypoperfusion may worsen the impact of successive ischemic intervals. Although levels of PCr and ATP indicate adequate recovery of energy metabolism between occlusions, slight elevations of brain lactate persist, consistent with continued hypoxia. Under the conditions employed in these studies, repeated occlusions give rise to progressively more prolonged deficits in brain protein synthesis activity, which may thus provide a useful index of the severity of the accumulated ischemic insult. Continued studies using this well-defined model should provide further insight into the pathophysiology of ischemic brain edema.

Observations on cerebral ischaemia in cats at injury threshold levels.

The potential for recovery of brain tissue subjected to ischaemia at a threshold level of injury was evaluated in cats subjected to 20 min middle cerebral artery occlusion. In addition to assessment of regional cerebral blood flow and water content, the permeability of the bloodbrain barrier and morphological changes detected by light microscopy were studied at various time intervals. Our observations revealed that although a similar reduction of blood flow during arterial occlusion was found both in the caudate nucleus and the cerebral cortex, the reactive hyperaemia was consistently higher in the caudate nucleus than in the cortex. After 24 h the caudate nucleus also revealed a significantly higher water content and increased vascular permeability than the cortex. Morphological observations at 24 h in areas affected by ischaemia showed widespread, marked ischaemic neuronal injury, whereas at 3 d there was, in addition, a vigorous proliferative reaction of vascular elements. Cats sacrificed at 14 d revealed a remarkably good preservation of neurons, both in the caudate nucleus and cortex which otherwise showed a few circumscribed, small, infarcts surrounded by normal nerve cells. Our study suggests that neurons injured at threshold level have a considerable capacity for recovery. Otherwise, with a similar degree of ischaemia, the caudate nucleus appears more prone to increased vascular permeability and oedema than the cerebral cortex.