Origin of functional de novo genes in humans from "hopeful monsters".

For a long time, it was believed that new genes arise only from modifications of preexisting genes, but the discovery of de novo protein-coding genes that originated from noncoding DNA regions demonstrates the existence of a "motherless" origination process for new genes. However, the features, distributions, expression profiles, and origin modes of these genes in humans seem to support the notion that their origin is not a purely "motherless" process; rather, these genes arise preferentially from genomic regions encoding preexisting precursors with gene-like features. In such a case, the gene loci are typically not brand new. In this short review, we will summarize the definition and features of human de novo genes and clarify their process of origination from ancestral non-coding genomic regions. In addition, we define the favored precursors, or "hopeful monsters," for the origin of de novo genes and present a discussion of the functional significance of these young genes in brain development and tumorigenesis in humans. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

PD-L1 expression, tumor mutational burden, and response to immunotherapy in patients with MET exon 14 altered lung cancers.

Background: MET exon 14 alterations are actionable oncogenic drivers. Durable responses to MET inhibitors are observed in patients with advanced MET exon 14-altered lung cancers in prospective trials. In contrast, the activity of immunotherapy, PD-L1 expression and tumor mutational burden (TMB) of these tumors and are not well characterized. Patients and methods: Patients with MET exon 14-altered lung cancers of any stage treated at two academic institutions were identified. A review of clinicopathologic and molecular features, and an analysis of response to single-agent or combination immune checkpoint inhibition were conducted. PD-L1 immunohistochemistry was carried out and TMB was calculated by estimation from targeted next-generation sequencing panels. Results: We identified 147 patients with MET exon 14-altered lung cancers. PD-L1 expression of 0%, 1%-49%, and ≥50% were 37%, 22%, and 41%, respectively, in 111 evaluable tumor samples. The median TMB of MET exon 14-altered lung cancers was lower than that of unselected non-small-cell lung cancers (NSCLCs) in both independently evaluated cohorts: 3.8 versus 5.7 mutations/megabase (P < 0.001, n = 78 versus 1769, cohort A), and 7.3 versus 11.8 mutations/megabase (P < 0.001, n = 62 versus 1100, cohort B). There was no association between PD-L1 expression and TMB (Spearman's rho=0.18, P = 0.069). In response-evaluable patients (n = 24), the objective response rate was 17% (95% CI 6% to 36%) and the median progression-free survival was 1.9 months (95% CI 1.7-2.7). Responses were not enriched in tumors with PD-L1 expression ≥50% nor high TMB. Conclusion: A substantial proportion of MET exon 14-altered lung cancers express PD-L1, but the median TMB is lower compared with unselected NSCLCs. Occasional responses to PD-1 blockade can be achieved, but overall clinical efficacy is modest.

Use of PRO Measures to Inform Tolerability in Oncology Trials: Implications for Clinical Review, IND Safety Reporting, and Clinical Site Inspections.

Cancer therapeutics frequently lead to symptomatic adverse events (AE) that can affect treatment tolerability. The NCI has developed the Patient-Reported Outcomes version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE) to assess symptomatic AEs by direct patient self-report. Although longitudinal assessment of patient-reported symptomatic AEs holds promise to better inform treatment tolerability, using patient-reported outcome (PRO) measures to assess symptomatic AEs has raised several regulatory and good clinical practice issues among those who conduct cancer clinical trials. These include concerns regarding trial monitoring, clinical review of PRO results by investigators and delegated clinical staff, whether PRO data on symptomatic AEs require investigational new drug (IND) safety reporting, and how the trial conduct and resultant PRO data will be assessed during clinical investigator site inspections. This article addresses current thinking regarding these issues in cancer clinical trials from the FDA, the NCI, and the Office for Human Research Protections. PRO measures, such as PRO-CTCAE, that assess symptomatic AEs in cancer trials are considered similar to other PRO assessments of symptoms, function, and health-related quality of life and can generate complementary data that may inform tolerability. Clarity on operational concerns related to incorporating PRO measures to inform tolerability is critical to continue the advancement of rigorous PRO assessment in cancer clinical trials. Clin Cancer Res; 24(8); 1780-4. ©2017 AACRSee related commentary by Nipp and Temel, p. 1777.

Antibody-mediated thyroid dysfunction during T-cell checkpoint blockade in patients with non-small-cell lung cancer.

Background: Programmed cell death protein-1 (PD-1) blockade therapies have demonstrated durable responses and prolonged survival in a variety of malignancies. Treatment is generally well tolerated although immune-related adverse events (irAEs) can occur. Autoimmune thyroid dysfunction is among the most common irAE, but an assessment of the clinical, mechanistic, and immunologic features has not been previously described. Patient and methods: Patients with advanced non-small-cell lung cancer (NSCLC) treated with pembrolizumab at Memorial Sloan Kettering Cancer Center (n = 51) as part of KEYNOTE-001 (NCT01295827) were included. Thyroid function test and anti-thyroid antibodies were assessed prospectively at each study visit, beginning before the first treatment. Frequency of development of thyroid dysfunction, association with anti-thyroid antibodies, clinical course, and relationship with progression-free survival and overall survival to treatment with pembrolizumab was evaluated. Results: Of 51 patients treated, 3 were hypothyroid and 48 were not at baseline. Ten of 48 [21%, 95% confidence interval (CI) 10% to 35%] patients developed thyroid dysfunction requiring thyroid replacement. Anti-thyroid antibodies were present in 8 of 10 patients who developed thyroid dysfunction, compared with 3 of 38 who did not (80% versus 8%, P < 0.0001). Thyroid dysfunction occurred early (median, 42 days) in the pembrolizumab course, and a majority (6 of 10 patients) experienced brief, transient hyperthyroidism preceding the onset of hypothyroidism; no persistent hyperthyroidism occurred. Both hyperthyroidism and hypothyroidism were largely asymptomatic. Overall survival with pembrolizumab was significantly longer in subjects who developed thyroid dysfunction (hazard ratio, 0.29; 95% CI 0.09-0.94; P = 0.04). Conclusions: Thyroid dysfunction during pembrolizumab treatment of NSCLC is common and is characterized by early-onset, frequently preceded by transient hyperthyroidism, closely associated with anti-thyroid antibodies, and may be associated with improved outcomes. The presence of antibody-mediated toxicity in T-cell-directed therapy suggests an under-recognized impact of PD-1 biology in modulating humoral immunity.

Sensitizing acute myeloid leukemia cells to induced differentiation by inhibiting the RIP1/RIP3 pathway.

Tumor necrosis factor-α (TNF-α)-induced RIP1/RIP3 (receptor-interacting protein kinase 1/receptor-interacting protein kinase 3)-mediated necroptosis has been proposed as an alternative strategy for treating apoptosis-resistant leukemia. However, we found that most acute myeloid leukemia (AML) cells, especially M4 and M5 subtypes, produce TNF and show basal level activation of RIP1/RIP3/MLKL signaling, yet do not undergo necroptosis. TNF, through RIP1/RIP3 signaling, prevents degradation of SOCS1, a key negative regulator of interferon-γ (IFN-γ) signaling. Using both pharmacologic and genetic assays, we show here that inactivation of RIP1/RIP3 resulted in reduction of SOCS1 protein levels and partial differentiation of AML cells. AML cells with inactivated RIP1/RIP3 signaling show increased sensitivity to IFN-γ-induced differentiation. RIP1/RIP3 inactivation combined with IFN-γ treatment significantly attenuated the clonogenic capacity of both primary AML cells and AML cell lines. This combination treatment also compromised the leukemogenic ability of murine AML cells in vivo. Our studies suggest that inhibition of RIP1/RIP3-mediated necroptotic signaling might be a novel strategy for the treatment of AML when combined with other differentiation inducers.

A phase II study of axitinib (AG-013736) in patients with incurable adenoid cystic carcinoma.

BACKGROUND: Recurrent/metastatic adenoid cystic carcinoma (ACC) is an incurable disease with no standard treatments. The majority of ACCs express the oncogenic transcription factor MYB (also c-myb), often in the context of a MYB gene rearrangement. This phase II trial of the tyrosine kinase inhibitor (TKI) axitinib (Pfizer) tested the hypothesis that targeting pathways activated by MYB can be therapeutically effective for ACC. PATIENTS AND METHODS: This is a minimax two-stage, phase II trial that enrolled patients with incurable ACC of any primary site. Progressive or symptomatic disease was required. Patients were treated with axitinib 5 mg oral twice daily; dose escalation was allowed. The primary end point was best overall response (BOR). An exploratory analysis correlating biomarkers to drug benefit was conducted, including next-generation sequencing (NGS) in 11 patients. RESULTS: Thirty-three patients were registered and evaluable for response. Fifteen patients had the axitinib dose increased. Tumor shrinkage was achieved in 22 (66.7%); 3 (9.1%) had confirmed partial responses. Twenty-five (75.8%) patients had stable disease, 10 of whom had disease stability for >6 months. The median progression-free survival (PFS) was 5.7 months (range 0.92-21.8 months). Grade 3 axitinib-related toxicities included hypertension, oral pain and fatigue. A trend toward superior PFS was noted with the MYB/NFIB rearrangement, although this was not statistically significant. NGS revealed three tumors with 4q12 amplification, producing increased copies of axitinib-targeted genes PDGFR/KDR/KIT. Two 4q12 amplified patients achieved stable disease for >6 months, including one with significant tumor reduction and the longest PFS on study (21.8 months). CONCLUSIONS: Although the primary end point was not met, axitinib exhibited clinical activity with tumor shrinkage achieved in the majority of patients with progressive disease before trial enrollment. Analysis of MYB biomarkers and genomic profiling suggests the hypothesis that 4q12 amplified ACCs are a disease subset that benefit from TKI therapy.

Skeletal findings in children recently initiating glucocorticoids for the treatment of nephrotic syndrome.

SUMMARY: Eighty children with nephrotic syndrome underwent lumbar spine densitometry and vertebral morphometry soon after glucocorticoid initiation. We found an inverse relationship between glucocorticoid exposure and spine areal bone mineral density (BMD) Z-score and a low rate of vertebral deformities (8%). INTRODUCTION: Vertebral fractures are an under-recognized complication of childhood glucocorticoid-treated illnesses. Our goal was to study the relationships among glucocorticoid exposure, lumbar spine areal BMD (LS BMD), and vertebral shape in glucocorticoid-treated children with new-onset nephrotic syndrome. METHODS: Lateral thoracolumbar spine radiography and LS BMD were performed in 80 children with nephrotic syndrome (median age 4.4 years; 46 boys) within the first 37 days of glucocorticoid therapy. Genant semiquantitative grading was used as the primary method for vertebral morphometry; the algorithm-based qualitative (ABQ) method was used for secondary vertebral deformity analysis. RESULTS: Six of the 78 children with usable radiographs (8%; 95% confidence interval 4 to 16%) manifested a single Genant grade 1 deformity each. All deformities were mild anterior wedging (two at each of T6, T7, and T8). Four of the 78 children (5%; 95% confidence interval 2 to 13%) showed one ABQ sign of fracture each (loss of endplate parallelism; two children at T6 and two at T8). Two of the children with ABQ signs also had a Genant grade 1 deformity in the same vertebral body. None of the children with a Genant or ABQ deformity reported back pain. An inverse relationship was identified between LS BMD Z-score and glucocorticoid exposure. CONCLUSIONS: Although we identified an inverse relationship between steroid exposure and LS BMD soon after glucocorticoid initiation for childhood nephrotic syndrome, there was only a low rate of vertebral deformities. The clinical significance of these findings requires further study.

Prophylaxis of deep-vein thrombosis in fractures below the knee: a prospective randomised controlled trial.

The incidence of deep-vein thrombosis and the need for thromboprophylaxis following isolated trauma below the knee is uncertain. We have investigated this with a prospective randomised double-blind controlled trial using low molecular weight heparin with saline injection as placebo in patients aged between 18 and 75 years who had sustained an isolated fracture below the knee which required operative fixation. All patients had surgery within 48 hours of injury and were randomised to receive either the placebo or low molecular weight heparin for 14 days, after which they underwent bilateral lower limb venography, interpreted by three independent radiologists. Further follow-up was undertaken at two, six, eight and 12 weeks. A total of 238 patients fulfilled all the inclusion criteria, with 127 in the low molecular weight heparin group and 111 in the placebo group, all of whom underwent bilateral venography. There was no statistically significant difference in the incidence of deep-vein thrombosis between those patients treated with low molecular weight heparin or the placebo (p = 0.22). The number of deep-vein thromboses in the two groups was 11 (8.7%) and 14 (12.6%), respectively. Age and the type of fracture were significantly associated with the rate of deep-vein thrombosis (p = 0.001 and p = 0.009, respectively) but gender, comorbidities and the body mass index were not. The overall incidence of deep-vein thrombosis in this series was 11%. There was no clinical or statistical significant reduction in the incidence of deep-vein thrombosis with the use of thromboprophylaxis. However, we accept that owing to a cessation of funding, recruitment to this trial had to be ended prior to establishing the necessary sample size. Our results cannot, therefore, categorically exclude the possibility that low molecular weight heparin treatment could be beneficial. We recommend a further multicentre trial be undertaken to resolve this matter.

Transcription factor nuclear factor kappaB regulates the inducible expression of the human B1 receptor gene in inflammation.

Expression of the bradykinin B1 receptor gene is up-regulated in vascular smooth muscle cells (VSMCs) in response to a variety of inflammatory stimuli. We isolated the 5'-flanking region of the human bradykinin B1 receptor gene and examined its promoter activity by transient transfection analysis. This region (-2582 to +34) showed promoter activity inducible by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), and interleukin-1beta (IL-1beta) in VSMCs. Further deletion analysis revealed that constructs containing 111 base pairs of 5'-flanking sequence were sufficient for transcriptional induction. Mutagenesis of a nuclear factor kappaB (NF-kappaB)-like site at -64 to -55 abolished most of the LPS, TNF-alpha, and IL-1beta inducibility, whereas a mutation of a cyclic AMP response element at -50 to -43 markedly reduced the basal promoter activity, and a mutation of the activator protein 1 (AP-1) site at -78 to -72 had minimal effects. Nuclear extracts from LPS, TNF-alpha, and IL-1beta-treated VSMCs, IL-1beta-treated human hepatoma HepG2, and human lung fibroblast IMR-90 cells showed strong inducible binding activity to the NF-kappaB-like site by gel shift assays. These results demonstrated that NF-kappaB-like nuclear factor was involved in the inducible expression of the human bradykinin B1 receptor gene during inflammatory processes.

[Species-specific monoclonal antibodies against the major outer membrane protein (MOMP) of Chlamydia trachomatis].

The synthesized one quarter N-terminal MOMP of C. trachomatis was used for primary immunization of three male BALB/c mice (8 weeks of age), and the boost with C. trachomatis L1/440/Bu elementary bodies (EBs) was followed on day 14. Spleen cells from one mouse with good response of immunization were fused with murine myeloma NS-1 cells on day 24. The hybrid cell suspension was seeded into the wells of 96-well microtest plates which contained macrophage feeder layers. Anti-chlamydial antibodies in culture fluids were screened by ELISA with 1/4 MOMP & L1 EBs coated 96-well trays. Positive wells were cloned by limiting dilution. Four clones which secreted immunoglobulin G1 & G2a class were obtained after elimination of those clones that produced antibodies to C. psittaci strain EAE, C. pneumoniae strain ATCC VR1310 and uninfected BGMK cells. In micro-IF test, we found that the all four clones of MAbs reacted with our laboratory prepared L1, L2, A, B, C, E EBs, L2 tissue culture inclusions, as well as the EBs of all 15 standard serovars of C. trachomatis. The titers of their ascites were more than 1:12,800 in micro-IF test. It was shown that the four clones of MAbs reacted predominantly with 40,000 MOMP of C. trachomatis L1 in Western blot.

[Species-specific monoclonal antibodies against Chlamydia pneumoniae].

The purified elementary bodies (EBs) of C. pneumoniae ATCC VR1310 were used for primary immunization of male BALB/c mice (8 weeks of age), with the boost following on day 14. Spleen cells were fused with murine myeloma NS-1 cells on day 24 and hybrid cells were cloned by limiting dilution. Two clones, P17C2 & P33C, secreting species-specific monoclonal antibodies (MAbs) and immunoglobulin IgG2a class were obtained after elimination of those clones that produced antibodies against C. trachomatis L1, L2, A, B, C, E serovars, C. psittaci EAE strain and uninfected BGMK cell antigens. It was showed that the two clones of MAbs reacted with the MOMP 39.5 x 10(3) Da major outer membrane protein of all chlamydia species in Western blot and their ascites titers were more than 1:25 600 in Micro-IF test.

Interaction of calmodulin with synthetic deletion peptides of melittin.

The 26-residue peptide melittin present in bee venom has been shown to bind calmodulin tightly. In this study we synthesized the following series of deletion peptides of melittin by the solid-phase method: Mel12, Mel13, Mel14, Mel15, Mel15F. The results of this study show that the deletion peptides Mel14 and Mel15 have almost the same binding activity as the intact native peptide. Each deletion peptide forms a 1:1 complex with calmodulin according to electrophoresis analysis. When the tryptophanyl residue of Mel15 was replaced by the phenylalaninyl residue, the dissociation constant of the peptide-calmodulin complex increased. This shows the importance of the tryptophanyl residue for binding to calmodulin.