Pemetrexed plus platinum with or without pembrolizumab in patients with previously untreated metastatic nonsquamous NSCLC: protocol-specified final analysis from KEYNOTE-189.

BACKGROUND: In the phase III KEYNOTE-189 study (NCT02578680), pembrolizumab plus pemetrexed and platinum-based chemotherapy (pemetrexed-platinum) significantly improved overall survival (OS) and progression-free survival (PFS) in patients with previously untreated metastatic nonsquamous non-small-cell lung cancer (NSCLC) versus placebo plus pemetrexed-platinum. We report updated efficacy outcomes from the protocol-specified final analysis, including outcomes in patients who crossed over to pembrolizumab from pemetrexed-platinum and in patients who completed 35 cycles (∼2 years) of pembrolizumab. PATIENTS AND METHODS: Eligible patients were randomized 2 : 1 to receive pembrolizumab 200 mg (n = 410) or placebo (n = 206) every 3 weeks (for up to 35 cycles, ∼2 years) plus four cycles of pemetrexed (500 mg/m2) and investigators' choice of cisplatin (75 mg/m2) or carboplatin (area under the curve 5 mg·min/ml) every 3 weeks, followed by pemetrexed until progression. Patients assigned to placebo plus pemetrexed-platinum could cross over to pembrolizumab upon progression if eligibility criteria were met. The primary endpoints were OS and PFS. RESULTS: After a median follow-up of 31.0 months, pembrolizumab plus pemetrexed-platinum continued to improve OS [hazard ratio (HR), 0.56; 95% confidence interval (CI), 0.46-0.69] and PFS (HR, 0.49; 95% CI, 0.41-0.59) over placebo plus pemetrexed-platinum regardless of programmed death-ligand 1 expression. Objective response rate (ORR) (48.3% versus 19.9%) and time to second/subsequent tumor progression on next-line treatment (PFS2; HR, 0.50; 95% CI, 0.41-0.61) were improved in patients who received pembrolizumab plus pemetrexed-platinum. Eighty-four patients (40.8%) from the placebo plus pemetrexed-platinum group crossed over to pembrolizumab on-study. Grade 3-5 adverse events occurred in 72.1% of patients receiving pembrolizumab plus pemetrexed-platinum and 66.8% of patients receiving placebo plus pemetrexed-platinum. Fifty-six patients completed 35 cycles (∼2 years) of pembrolizumab; ORR was 85.7% and 53 (94.6%) were alive at data cut-off. CONCLUSIONS: Pembrolizumab plus pemetrexed-platinum continued to show improved efficacy outcomes compared with placebo plus pemetrexed-platinum, with manageable toxicity. These findings support first-line pembrolizumab plus pemetrexed-platinum in patients with previously untreated metastatic nonsquamous NSCLC.

Monitoring of somatic mutations in circulating cell-free DNA by digital PCR and next-generation sequencing during afatinib treatment in patients with lung adenocarcinoma positive for EGFR activating mutations.

Background: Analysis of circulating cell-free DNA (cfDNA) is under intensive investigation for its potential to identify tumor somatic mutations. We have now explored the usefulness of such liquid biopsy testing with both the digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) during treatment of patients with the epidermal growth factor receptor (EGFR) inhibitor afatinib. Patients and methods: Eligible patients had advanced lung adenocarcinoma with EGFR activating mutations and were treated with afatinib. Plasma samples were collected before and during (4 and 24 weeks) afatinib treatment as well as at disease progression. Tumor and plasma DNA were analyzed by dPCR and NGS. Results: Thirty-five patients were enrolled. The objective response rate and median progression-free survival (PFS) were 77.1% and 13.8 months, respectively. Tumor and plasma DNA were available for 32 patients. dPCR and NGS detected EGFR activating mutations in 81.3% and 71.9% of baseline cfDNA samples, respectively. In 19 patients treated with afatinib for ≥24 weeks, the number of EGFR mutant alleles detected in cfDNA by dPCR declined rapidly and markedly after treatment onset, becoming undetectable or detectable at only a low copy number (<10 copies per milliliter) at 4 weeks. Median PFS was slightly longer for patients with undetectable EGFR mutant alleles in cfDNA at 4 weeks than for those in whom such alleles were detectable (14.3 versus 10.0 months). A total of 45 somatic mutations was identified in baseline tumor DNA, and 30 (66.7%) of these mutations were identified in cfDNA by NGS. Allele frequency for somatic mutations in cfDNA determined by NGS changed concordantly during afatinib treatment with the number of EGFR mutant alleles determined by dPCR. Conclusions: Monitoring of cfDNA by dPCR is informative for prediction of afatinib efficacy, whereas that by NGS is reliable and has the potential to identify mechanisms of treatment resistance.

Viral load and ganciclovir (GCV) concentration in cerebrospinal fluid of patients successfully treated with GCV or valGCV for human herpesvirus 6 encephalitis/myelitis following umbilical cord blood transplantation.

We describe successful treatment of 3 cases of human herpesvirus 6 (HHV-6) encephalitis/myelitis following cord blood transplantation (CBT). Ganciclovir (GCV) (10 mg/kg/day) reduced HHV-6 load to undetectable levels in cerebrospinal fluid (CSF). Early dose reduction in the presence of HHV-6 detectable in CSF resulted in an increased HHV-6 load. GCV was capably shifted to valganciclovir (VGCV) with an almost equivalent concentration. GCV/VGCV may be effective for HHV-6 encephalitis/myelitis after CBT, although HHV-6 load in CSF should be monitored.

A phase I dose-escalation study of eribulin and S-1 for metastatic breast cancer.

BACKGROUND: We evaluated the safety, maximum-tolerated dose (MTD), pharmacokinetics, recommended dose for phase II (P2RD), and preliminary anticancer activity of a combination eribulin and S-1 therapeutic in metastatic breast cancer patients pretreated with anthracycline and taxane. METHOD: Patients aged 20-74 years were recruited. In level 1, patients received S-1 (65 mg m(-2)) from day 1 to 14, and eribulin (1.1 mg m(-2)) on day 1 and 8 in a 21-day cycle. In level 2, eribulin was increased to 1.4 mg m(-2). In level 3, S-1 was increased to 80 mg m(-2). RESULTS: Twelve patients were enrolled into three cohorts. Planned dose escalation was completed, with one case exhibiting dose-limiting toxicity (grade 3 hypokalaemia) at level 3, without reaching the MTD. The P2RD was determined to be level 2 (eribulin 1.4 mg m(-2) and S-1 65 mg m(-2)). The most common grade 3 or 4 toxicity was neutropenia (83.3%), followed by febrile neutropenia (25.0%). Five of eleven patients (41.7%) with measurable disease had a partial response. Pharmacokinetics were characterised by dose-dependent elimination and nonlinear exposure. CONCLUSION: Dose level 3 was not tolerated owing to febrile neutropenia development. Thus, intermediate dose level 2 was recommended for further evaluation. Preliminary antitumour activity warrants further investigation in this setting.

In vitro expansion of CD34(+)CD38(-) cells under stimulation with hematopoietic growth factors on AGM-S3 cells in juvenile myelomonocytic leukemia.

Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34(+) cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34(+) cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34(+) cells on AGM-S3 cells. The expansion potential of CD34(+) cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34(+) cells were negative for CD38 and cryopreservable. Cultured JMML CD34(+)CD38(-) cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34(+) cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.

An open-label, dose-escalation, safety, and pharmacokinetics phase I study of ombrabulin, a vascular disrupting agent, administered as a 30-min intravenous infusion every 3 weeks in Japanese patients with advanced solid tumors.

PURPOSE: To determine ombrabulin's maximum tolerated dose and dose recommended for Japanese patients with advanced solid tumors and to assess its antitumor activity and overall safety and pharmacokinetic profiles. METHODS: This was a multi-center, open-label, sequential-cohort, dose-escalation phase I study of ombrabulin, a vascular disrupting agent, administered once every 3 weeks. Patients were treated with 15.5, 25, 35, or 50 mg/m(2) ombrabulin over a 30-min intravenous infusion. The recommended dose was the highest dose at which <33 % of all evaluable patients experienced dose-limiting toxicities (DLTs) during the first treatment cycle or 50 mg/m(2) (recommended in Caucasian patients) if the previous definition was not met. RESULTS: Fifteen patients were treated. No DLT occurred with 15.5, 25, or 35 mg/m(2) ombrabulin. In the 50 mg/m(2) group, one patient had Grade 3 lymphopenia, and another experienced Grade 2 hypertension and Grade 3 diarrhea judged as DLTs. The most frequent related adverse events in this group were diarrhea, nausea, and hypertension. Two patients had Grade 3 anemia, one at the 15.5 mg/m(2) and the other at the 50 mg/m(2). No AEs necessitating dose reduction or Grade 4 AEs were observed. Overall, five patients had stable disease. Pharmacokinetic parameters were comparable to those in non-Japanese patients. CONCLUSIONS: Ombrabulin treatment once every 3 weeks was well tolerated in Japanese patients with advanced solid tumors. The dose recommended is 50 mg/m(2), as in Caucasian patients. The safety and pharmacokinetic profiles were comparable between Japanese and Caucasian patients (funded by Sanofi; ClinicalTrials.gov number, NCT00968916).

Is response rate increment obtained by molecular targeted agents related to survival benefit in the phase III trials of advanced cancer?

BACKGROUND: It remains unclear whether response rate (RR) is related to survival benefit in phase III trials of advanced cancer treated with molecular targeted agents (MTA) in combination with standard therapies. MATERIALS AND METHODS: We carried out a systematic search of PubMed for randomized phase III trials of four solid tumors examining the efficacy of MTA when added to a standard therapy. We examined whether there were any associations between RR increment obtained by the addition of targeted agents (DeltaRR) and survival benefit in phase III trials. RESULTS: We identified 26 phase III trials of MTA with a total of 21 156 patients and 29 experimental arms of MTA. Studies which showed significant survival benefit had higher DeltaRR compared with those which did not show significant benefit. In the receiver operating characteristic curve analysis, using a 7% gain as threshold value for DeltaRR allowed assessment of survival benefit with high sensitivity and specificity. There were also significant relationships between DeltaRR and hazard ratios for overall survival and progression-free survival in the linear regression analysis. CONCLUSION: RR increment obtained by the addition of MTA to a standard therapy may be useful to predict survival benefit in clinical phase III trials of advanced cancer.

Novel real-time monitoring system for human cytomegalovirus-infected cells in vitro that uses a green fluorescent protein-PML-expressing cell line.

Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for "immediate-early 1") protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

In vitro and in vivo analysis of human herpesvirus-6 U90 protein expression.

In order to establish a reliable method for the detection of human herpesvirus-6 (HHV-6) B antigens in peripheral blood mononuclear cells (PBMCs) collected from HHV-6 infected patients, we created a polyclonal antibody against the HHV-6 B U90 protein (IEA/ex3) and used it to examine the expression of this protein in virus-infected cells and patients' PBMCs. This antibody reacted with 170 and 195 kDa proteins in HHV-6 B-infected cord blood mononuclear cells. The IEA/ex3 antigen was detected in cord blood mononuclear cells at 6 hr post-infection, and the number of infected cells reached its maximum at 48 hr post-infection. The antigen stained in a punctate pattern and partially localized to the promyelocytic leukemia (PML) protein body. We also examined 60 PBMC samples from 60 febrile children (3-19 months old) and detected IEA/ex3 antigen in the PBMCs by laser-scanning microscopy. HHV-6 was isolated from 31 of the 60 samples. The sensitivity and specificity of the antigen detection were 84% (26/31) and 97% (28/29), respectively, in the samples with virus detected. The mean number of antigen-positive PBMCs was 409/10(6) cells in 20 samples with viral isolation. A significant correlation (r = 0.566; P = 0.008) was observed between the viral load and number of antigen-positive cells. Although IEA/ex3 antigen was detected by laser-scanning microscopy in PBMCs (without cultivation) collected from six patients with isolated virus, it was detected in only one sample by conventional fluorescence microscopy. Increasing the intensity by cultivation (24 hr) resulted in a higher detection rate (5/6) even by conventional fluorescence microscopy, which is available in most hospital laboratories.

Combination phase I study of nedaplatin and gemcitabine for advanced non-small-cell lung cancer.

To establish the toxicities and maximum tolerated dose (MTD) of nedaplatin with gemcitabine, and to observe their antitumour activity, we conducted a combination phase I study in advanced non-small-cell lung cancer (NSCLC). Patients received nedaplatin (60-100 mg m(-2) given intravenously over 90 min) on day 1, and gemcitabine (800-1000 mg m(-2) given intravenously over 30 min) on days 1, 8, every 3 weeks. In total, 20 patients with locally advanced or metastatic NSCLC who received no prior chemotherapy or one previous chemotherapy regimen were enrolled. The most frequent toxicities were neutropenia and thrombocytopenia; nonhaematological toxicities were generally mild. Three out of six patients experienced dose-limiting toxicities (neutropenia, thrombocytopenia and delayed anaemia) at dose level 4, 100 mg m(-2) nedaplatin with 1000 mg m(-2) gemcitabine, which was regarded as the MTD. There were three partial responses, for an overall response rate of 16.7%. The median survival time and 1-year survival rate were 9.1 months and 34.1%, respectively. This combination is well tolerated and active for advanced NSCLC. The recommended dose is 80 mg m(-2) nedaplatin with 1000 mg m(-2) gemcitabine. This combination chemotherapy warrants a phase II study and further evaluation in prospective randomised trials with cisplatin- or carboplatin-based combinations as first-line chemotherapy for advanced NSCLC.

Oncolytic viral therapy using a spontaneously generated herpes simplex virus type 1 variant for disseminated peritoneal tumor in immunocompetent mice.

The present study demonstrates that a clonal derivative (HF10) of HSV-1 strain HF effectively treated disseminated peritoneal neoplasm in an immunocompetent animal model and that all of survived mice acquired resistance to rechallenge with tumor cells. The survival time of mice treated with HF10 was longer than that of mice treated with hrR3, indicating that the oncolytic effect of HF10 was more potent than that of hrR3 in this animal model. HF10 induces syncytia formation in vitro, whereas hrR3 forms rounded CPE. The sequential administration of HF10 gave a long term survival of more than 90 days after tumor injection, with no signs of disease, in 8 of the 9 treated mice. The results suggest that treatment of disseminated peritoneal tumor with HF10 induces a specific antitumor immune response. Genomic structure determination showed that HF10 has a deletion of 3.9-kilobase pair (kbp) in the right end of UL and UL/IRL junction, resulting in the loss of UL 56 expression. A 2.3 kbp deletion and extensive rearrangement were also observed in the left end of the genome.

An endoplasmic reticulum protein, p180, is highly expressed in human cytomegalovirus-permissive cells and interacts with the tegument protein encoded by UL48.

We have used a virus overlay assay to detect cellular proteins associated with human cytomegalovirus (HCMV) particles. The radiolabeled HCMV particles specifically bound to two host proteins with molecular sizes of 150 and 180 kDa. By a micro-amino-acid sequencing technique, the 180-kDa protein was identified as a human homologue of the ES130/p180 ribosome receptor (p180), which is an integral endoplasmic reticulum (ER) membrane protein possessing a very unique tandem repeat domain at its N-terminal region. The virus overlay assay using truncated p180 polypeptides revealed that HCMV binding to human p180 occurred through the N-terminal region. In HCMV-permissive cells the high level of expression of the human p180 protein was clearly observed regardless of cell type. Furthermore, we showed that p180 binds to the UL48 gene product, which is one of the predominant tegument proteins of HCMV and which is considered to be tightly associated with the capsid. The interaction between the two proteins was assumed to be specific and was observed both in vitro and in vivo. During the late phase of infection, the unique relocation of human p180 was observed, that is, to the juxtanuclear region, which appeared to be in the vicinity of the area where naked virions were frequently observed in an electron-microscopic study. Thus our data suggest that p180 interacts with the HCMV tegument, at least through pUL48, during the HCMV replication process. We discuss the possible role of the interaction between p180 and pUL48 in the intracellular transport of HCMV virions.

Spontaneous reduction in Epstein-Barr virus (EBV) DNA copy number in EBV-infected epithelial cell lines.

We found that spontaneous and 12-0-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus (EBV) reactivation occurred in short-term (ST)-cultured EBV-infected epithelial cell lines GT38 and GT39 after their establishment; however, it diminished in the long-term (LT)-cultured cells passaged for more than 2 years from ST-cultured cells. We hypothesized that the EBV reactivation may be related to the EBV DNA copy number in the cells. A higher level of EBV DNA content was detected in ST-cultured cells than in LT-cultured cells by Southern hybridization using an EBV DNA XhoI probe. Fluorescence in situ hybridization using EBV DNA BamHI W fragments showed that ST-cultured cells contained a higher EBV DNA copy number than that of LT-cultured cells. EBV DNA-negative cells were detected in small proportions in LT-cultured cells, but were undetected in ST-cultured cells. These results demonstrate that EBV genomes are not maintained stably in the cell lines, and some of them are lost in continuous passages of the cells. We discuss the mechanisms of reduction of EBV reactivation and EBV DNA in the cell lines.

Effect of transforming growth factor-beta1 on the cell growth and Epstein-Barr virus reactivation in EBV-infected epithelial cell lines.

Transforming growth factor (TGF)-beta1 is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems. In this study, we found that gastric tissue-derived Epstein-Barr virus (EBV)-infected epithelial cell lines GT38 and GT39 had resistance to TGF-beta1-mediated growth inhibition and apoptosis compared to a TGF-beta1-susceptible gastric carcinoma cell line HSC-39. However, TGF-beta1 partially induced EBV reactivation in GT38 and GT39 cells, as shown by the induction of EBV immediate-early BZLF1 RNA and its protein product ZEBRA and early antigen-D. The expressions of TGF-beta receptor I and II were detected in GT38 and GT39 cells by Northern and Western blot analyses. Both cell lines spontaneously produced the TGF-beta1, which was sufficient for inhibiting cell growth of HSC-39 cells. Taken together, these data suggest that TGF-beta1 may be a key factor for EBV reactivation and selective growth of EBV-infected epithelial cells in vivo.

Effect of the deletion of US2 and US3 from herpes simplex virus type 2 on immune responses in the murine vagina following intravaginal infection.

We investigated the effects of US2 and US3 deficiencies of herpes simplex virus type 2 (HSV-2) on host immunity in a murine model of genital herpes infection. Viral clearance from the vaginal mucosa was more rapid in mice infected with a US3-deficient mutant L1BR1 as compared with a wild-type 186 or YY2 (US2-deficient mutant) infection, although there was no significant difference among them in initial growth in the early stage of infection. Flow cytometric studies revealed that the number of vaginal mononuclear cells in L1BR1-infected mice was significantly greater than that in 186- or YY2-infected mice. Dendritic cells, macrophages and T cells were induced more rapidly and in greater numbers within the vaginas of L1BR1-infected mice. Moreover, the levels of IL-12 and IFN-gamma increased in L1BR1-infected mice over levels in 186-infected mice. These results indicate that a US3 deficiency alters the induction of the host immune response; therefore, the inactivation of US3 may be a promising strategy in the development of novel vaccines for genital herpes.

Human-herpesvirus-8-encoded K8 protein colocalizes with the promyelocytic leukemia protein (PML) bodies and recruits p53 to the PML bodies.

Promyelocytic leukemia protein (PML) bodies are nuclear sites for both input viral genome deposition and immediate-early (IE) gene transcription during infection with certain human DNA viruses, such as human cytomegalovirus (HCMV), herpes simplex virus type 1, and adenovirus. In this study, we showed that the K8 (K-bZIP) protein, an early protein encoded by the human herpesvirus 8 (HHV-8), colocalized with the PML bodies in HHV-8-infected primary effusion lymphoma cells. Cotransfection of two plasmids expressing the K8 protein and green-fluorescence protein (GFP)-PML fusion protein into 293T cells revealed that the K8 protein colocalized with PML in cells with high PML expression. Overexpression of the K8 protein in Chinese hamster ovary (CHO) cells with stable GFP-PML expression did not induce the dispersion of the PML bodies, unlike the IE1 protein of HCMV. Transfection of a truncated K8 gene revealed that the leucine zipper domain of the K8 protein was required for the colocalization with PML. We also demonstrated that the K8 protein bound to p53 in vivo and in vitro, and that high expression of the K8 protein caused the accumulation of p53 to the PML bodies in CHO cells, suggesting that the K8 protein functions in the recruitment of p53 to the PML bodies. These data suggest that the K8 protein may be associated with the functional modulation of p53 in the nucleus during the lytic phase of HHV-8.

Varicella zoster virus antigens in the epidermis of patients with herpes zoster before and after treatment with acyclovir: an immunohistochemical study.

Using a monoclonal antibody to varicella zoster virus (VZV), an immunohistochemical study was performed before and after treatment with acyclovir (750 mg/day intravenously for 5 - 7 days) to investigate the distribution of VZV antigens in the epidermis of four in-patients with herpes zoster, and to correlate their presence with clinical manifestations of the disease. Biopsy specimens were obtained from epidermal lesions on admission to hospital prior to acyclovir administration, and again following treatment. In all cases, VZV antigens were found extensively in the erythematous and vesicular lesions before treatment, but they were not detected 5 - 7 days later in the ulcerative, crusted or pigmented lesions after acyclovir therapy. Further controlled studies will be necessary to compare the distribution of epidermal VZV antigens in acyclovir-treated patients with that in a placebo group to determine whether the loss of VZV antigens was due to acyclovir or to a natural decrease over time.