Complex DNA damage may manifest in double-strand breaks (DSBs) and non-DSB, bistranded, oxidatively induced clustered DNA lesions (OCDLs). Although the carcinogen benzoαpyrene (BαP) has been shown to ...induce chromosomal aberrations and transformation of mammary cells, it is not known whether this compound engenders clustered DNA damage. Normal primary breast tissue-derived cells were treated with BαP, and the levels of DNA lesions, chromosomal aberrations, total antioxidant capacity (TAC), and reactive oxygen species (ROS) were determined. DNA from cells treated with 2 and 8
µM BαP exhibited increases of 3- and 4-fold in APE1 (
p
<
0.001), 11- and 19-fold in Endo III (
p
<
0.001), and 8- and 15-fold in hOGG1 (
p
<
0.001) OCDLs, respectively, compared to the 0
µM BαP-treated (control) group. Mammary cells treated with 8
µM BαP produced 0.12 aberrations per cell (
p
<
0.05) and there was a strong positive correlation (
r
=
0.91) between the levels of OCDLs and those of chromosomal aberrations. Finally, TAC was decreased by 25% (
p
<
0.02), whereas ROS production increased by 2-fold (
p
<
0.02) in cells treated with 8
μM BαP compared to the control group. In conclusion, oxidatively induced clustered DNA damage mediated through differential expression of APE1, reduced TAC, and increased ROS may play a significant role in the chemically induced transformation of normal primary mammary cells.
Angiogenesis is critical for the growth and proliferation of tumors as well as for normal development. We now describe a novel role for histidine-rich glycoprotein (HRGP) in the modulation of ...angiogenesis. HRGP is a plasma protein that circulates in relatively high concentrations (1.5 microM), but has no known function in vivo. We have shown previously that HRGP binds with high affinity to thrombospondin-1 (TSP-1), a homotrimeric glycoprotein that is a potent inhibitor of angiogenesis. The antiangiogenic activity of TSP-1 is mediated by the binding of properdin-like type I repeats to the receptor CD36. We found that binding of HRGP to TSP-1 was similarly mediated by TSP type I repeats. HRGP colocalized with TSP-1 in the stroma of human breast cancer specimens, and this interaction masked the antiangiogenic epitope of TSP-1. In assays performed in vitro of endothelial cell migration and tube formation, and in vivo corneal angiogenesis assays, HRGP inhibited the antiangiogenic effect of TSP-1. These studies suggest that HRGP can modulate the antiangiogenic activity of TSP-1, and identify a potential mechanism of resistance to the antiangiogenic effect of TSP-1.
BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune disorder caused by antibodies directed at self-antigens leading to platelet destruction. Autoantibodies from most patients with ITP are ...directed to platelet membrane glycoprotein(GP) IIb-IIIa or GPIb-IX. 80% of ITP cases are classified as primary and it's unclear why auto reactive antibodies are formed in these patients. Studies from the United States, Asia and Europe show an increasing incidence of ITP with aging. Thrombopoietin receptor agonists (TPO-RA) interact directly with the TPO receptor on megakaryocytes to stimulate platelet production and can surprisingly produce sustained remissions of chronic ITP in some patients even after discontinuation of treatment. The heterogeneity of the course of ITP and the response to treatment, especially the response to treatment with thrombopoietin receptor agonists (TPO-RA), suggests pathogenic and clinical diversity of ITP. Clonal hematopoiesis is known to occur in elderly patients and has been associated with adverse outcomes including hematologic cancers and cardiovascular disease. We hypothesized that clonal hematopoiesis and associated passenger somatic mutations contribute to loss of immune tolerance in some ITP patients, causing the generation of auto-reactive antibodies and affecting the response to TPO-RA.
METHODS: Platelet RNA was extracted from 13 blood samples obtained from 11 ITP patients using Direct-zolTM RNA MicroPrep Kit (Zymo Research). 2 patients provided samples during both relapse and remission. cDNA generation was performed from enriched total RNA using SMARTerR Target RNA Capture for IlluminaR Kit (Clontech). Target-specific biotinylated DNA probes were designed to hybridize to mRNA transcripts for common platelet antigens including (GP) IIb-IIIa and GPIb-IX. cDNA libraries were prepared using the Nextera™ XT DNA Library preparation kit. Following the pooling of libraries they were sequenced on an Illumina Nextseq 500 instrument in Mid-output mode with single-ended reads of 150 bp.
RESULTS: In one of the paired patient specimens, we found substantial differences in platelet populations between relapse and remission on a TPO-RA. Different previously-reported non-disease-causing heterozygous single nucleotide variants (SNVs) were detected during relapse and remission. Expression at a level approximating 50% in remission and a reciprocal expression in relapse suggests that 2 point mutations leading to a missense amino acid change (Glu to Lys at amino acid 671) in GPIIb (ITGA2B) and (Thr to Met at amino acid 161) in GPIb (GP1BA) provoke an immune response (Table 1). Further, the reciprocal expression at these levels is consistent with expression of a somatic mutation in a clonal fashion.
CONCLUSION: We documented that unique platelet clones were expressed during relapse and remission of ITP. These findings demonstrate that clonal expression of platelets bearing somatic mutations in target glycoproteins are associated with ITP disease course and response to therapy. Such somatic mutations are likely to be passenger mutations acquired prior to clonal expansion. Expression of a somatic mutation in a clonal fashion may drive autoimmune response.
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No relevant conflicts of interest to declare.
Background: Immunotherapy directed against the PD-1/PDL-1 (programmed cell death protein ligand 1) pathway has shown activity across a range of malignancies. Immune-related adverse events have been ...shown to be associated with the efficacy of PD-1/PDL-1 inhibitors in patients with melanoma and non-small cell lung cancer. Thrombocytopenia has been reported in up to 33% of patients receiving anti PD-1/PDL-1 therapy. Our hypothesis is that immune-mediated thrombocytopenia may be a biomarker for response to anti PD-1/PDL-1 therapy.
Methods: We collected data on 250 patients aged 18 years and older who were treated with anti PD-1/PDL-1 therapy at the University of Oklahoma Health Sciences Center between January 2014 and January 2016. Initial platelet count at baseline as well as nadir platelet count during treatment were examined. Patient's clinicopathologic characteristics were analyzed using simple descriptive statistics median (range) for continuous covariates and n (%) for categorical variables. Kaplan Meier Analysis was performed to assess how thrombocytopenia was associated with overall survival (OS) and progression free survival (PFS). Patients were grouped according to their nadir platelet count: normal platelets (≥150k), grade 1 (75k-150k), and combined grades 2, 3, 4 (<75k). Cox proportional hazard regression models were used to assess the association of thrombocytopenia with OS and PFS adjusted for various covariates. The objective of the study is to assess whether the degree of thrombocytopenia correlates with the PFS and OS in patients receiving anti PD-1/PDL-1 therapy.
Results: A total of 250 patients were reviewed in this study. After excluding 27 patients with baseline platelet count <100k and 14 patients with missing platelet count data, 209 patients were included in the analysis. Fifty-five percent were females. Median age at diagnosis was 62 (23-91) years. The most common cancer diagnoses were lung cancer (21.1%), melanoma (17%), and ovarian cancer (9%). Nivolumab was the most commonly used agent (40.9%), followed by Pembrolizumab (38.4%). Median number of treatments received was 5. Median platelet count at baseline was 227 (102-535). Median nadir platelet count was 180 (4-552). Thrombocytopenia of any grade was observed in 70 (33%) patients .The number of patients with grade 1, 2, and 3/4 were 56 (26.8%), 1 (0.5%) and 13 (6.2%), respectively. Median follow-up time was 226 days. Univariate analysis showed that patients with grade I thrombocytopenia had higher PFS and OS when compared to patients who did not develop thrombocytopenia HR 0.48 (95% CI, 0.29-0.81), P = 0.0053 and HR 0.49 (95% CI, 0.27-0.9), P = 0.0209, respectively. When adjusted for age, cancer type, performance status, and line of treatment, grade 1 thrombocytopenia remained positively associated with survival outcome HR 0.50 (95% CI, 0.29-0.87), P = 0.0146 for PFS and HR 0.40 (95% CI, 0.21-0.77), P = 0.0061 for OS. Only 14 patients had grade 2-4 thrombocytopenia, and there was no statistically significant difference in survival compared to patients with no thrombocytopenia adjusted HR 0.52 (95% CI, 0.2-1.38), P = 0.189 for OS.
Conclusion: Our study shows that in patients treated with anti PD-1/PDL-1 therapy, grade 1 thrombocytopenia appears to be positively correlated with survival outcomes. This survival advantage was not seen with higher grades of thrombocytopenia. PD-L1 was recently shown to be expressed on human platelets; thus, thrombocytopenia is likely immune-mediated. Thrombocytopenia may be a biomarker for efficacy of immune checkpoint therapy. The predictive significance of thrombocytopenia in patients receiving PD-1/PDL-1 therapy warrants further investigations.
No relevant conflicts of interest to declare.
![Role of benzo[α]pyrene in g...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0891-5849/small "Role of benzo[α]pyrene in generation of clustered DNA damage in human breast tissue")
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