Two Mutation Model for Carcinogenesis: Relative Roles of Somatic Mutations and Cell Proliferation in Determining Risk

1990 ◽  
pp. 136-152 ◽  
Author(s):  
Suresh H. Moolgavkar ◽  
Georg Luebeck ◽  
Mathisca de Gunst
Keyword(s):  
Cell Proliferation ◽  
Somatic Mutations ◽  
Mutation Model

scholarly journals The Molecular Basis of Long First Remissions in Normal Karyotype AML Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3827-3827
Author(s):  
Francesca Ferraro ◽  
Christopher A Miller ◽  
Amy Abdalla ◽  
Nichole Helton ◽  
Nathan Salomonis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Somatic Mutations ◽  
Conflicts Of Interest ◽  
Normal Karyotype ◽  
High Dose ◽  
Intermediate Risk ◽  
The Mean ◽  
Cebpa Mutations

Currently, it is not clear why some patients with acute myeloid leukemia (AML) can be "cured" with chemotherapy alone; are they living with small amounts of disease that is held in check by immunologic (or other) mechanisms, or is their disease really eradicated? The percentage of cytogenetically normal AML patients who have long (>5 years) first remissions (LFRs) after chemotherapy alone is low (about 9.1% in patients <60 years and 1.6% in >60 years1). For this reason, most intermediate risk patients are offered allogeneic transplantation to decrease their risk for relapse. To better understand mechanisms of chemotherapy sensitivity in AML, we performed an analysis of the mutation landscape and persistence, using samples from 8 normal karyotype LFR patients (without CEBPA mutations) who received standard "7+3" induction and high dose cytarabine consolidation as their only therapy. The mean age at diagnosis was 43.5 years, and the mean follow up in first remission is 7.6 years; none of these patients has relapsed to date. For each case, we performed enhanced exome sequencing at diagnosis (235x coverage of the entire exome, and ~1008x coverage of recurrently mutated AML genes). Each case had at least one documented AML driver mutation, with a median of 29 somatic mutations in the exome space. We created probes for 225 mutations (mean 28 per case), and performed error-corrected sequencing (Haloplex) for all available remission samples. The mean depth of Haloplex coverage was 1607x, and each sample had at least one AML-specific mutation assayed, with a sensitivity of 1 cell in 1,750 (0.06%). 7/8 patients demonstrated complete clearance of all mutations in all remission samples tested, which was confirmed with digital droplet PCR for 5 cases, with a sensitivity of detection of 1 cell in 100,000. In one case, we detected a persistent ancestral clone harboring DNMT3AR882H, which can be associated with long first remissions for some patients2. Strikingly, the founding clone in all 8 cases had one or more somatic mutations in genes known to drive cell proliferation (e.g. MYC, FLT3, NRAS, PTPN11, Figure 1 top panel). These are usually subclonal mutations that occur late during leukemic progression, suggesting that the presence of a "proliferative hit" in the founding clone might be important for chemotherapy clearance of all the AML cells in a given patient. To support this hypothesis, we analyzed the mutational clearance of 82 AML cases with paired diagnosis and day 30 post-chemotherapy bone marrow samples. We observed that, whether present in the founding clone or in subclones, mutations in MYC, CEBPA, FLT3, NRAS, and PTPN11 cleared after induction chemotherapy in all samples, while other mutations were often persistent at day 30 (e.g. DNMT3A, IDH1, IDH2, NPM1, TET2; Figure 1 bottom panel). Compared to other published sequencing studies of AML, MYC and NRAS mutations were significantly enriched in this small cohort (MYC p= 0.002, and NRAS p= 0.034), with MYC enrichment being particularly striking (37.5% versus 1.8%). All MYC mutations were canonical single base substitutions occurring in the highly conserved MYC Box 2 domain at the N-terminus of MYC (p.P74Q or p.T73N). Overexpression of MYCP74Q in murine hematopoietic progenitors prolonged MYC half life (89 min vs. 44 min for wild type), and enhanced cytarabine sensitivity at all concentrations tested (range 10-1000 nM, p=0.0003), both in vitro and in a MYC-driven leukemia model in vivo. MYC expression measured with flow cytometry in the blasts of the LFR samples was significantly higher (p=0.045) compared to unfavorable risk (complex karyotype) or other intermediate risk categories, but similar to good risk AML (biallelic CEBPA mutations, core binding factor fusion-associated AML, and AML with isolated NPMc), suggesting that activation of the MYC pathway may represent a shared feature of chemosensitive patients. Taken together, these data suggest that some intermediate patients who are effectively "cured" with chemotherapy alone may not have persistent subclinical disease, nor retained ancestral clones that could potentially contribute to relapse. Importantly, these patients often have mutations driving cell proliferation in the founding clone, indicating that the presence of specific mutations in all malignant cells may be critical for complete AML cell clearance with chemotherapy. 1. Blood Adv. 2018 Jul 10; 2(13): 1645-1650 2. N Engl J Med 2018; 378:1189-1199 Disclosures No relevant conflicts of interest to declare.

Somatic Activating Mutations In CXCR4 Are Common In Patients With Waldenstrom’s Macroglobulinemia, and Their Expression In WM Cells Promotes Resistance To Ibrutinib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4424-4424 ◽  
Author(s):  
Yang Cao ◽  
Zachary Hunter ◽  
Xia Liu ◽  
Lian Xu ◽  
Guang Yang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Somatic Mutation ◽  
Sanger Sequencing ◽  
Somatic Mutations ◽  
Akt Signaling ◽  
Wild Type ◽  
Control Vector ◽  
Whim Syndrome ◽  
Frame Shift

Background Waldenstrom's macroglobulinemia (WM) is an indolent non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells in the bone marrow. CXCR4 is a chemokine receptor that promotes the survival, migration, and adhesion to the bone marrow stroma of WM lymphoplasmacytic cells (LPC) through interactions with its ligand CXCL12. Through whole genome sequencing, we identified somatic mutations in CXCR4 that affected 1/3 of WM patients. These mutations were identical or functionally similar to those associated with Warts, Hypogammaglobulinemia, Infection, and Myelokathexis (WHIM) syndrome (Hunter et al, ASCO 2012), a rare autosomal dominant genetic disorder that is caused by frame shift or nonsense mutations in the carboxyl-terminal cytoplasmic tail of CXCR4. In WHIM syndrome, loss of the c-terminal tail of CXCR4 impairs receptor internalization, thereby prolonging G-protein and β-arrestin signaling (Lugane et al, Blood 2008). Ibrutinib induces WM cell death, and is highly active in WM (Treon et al, ICML-12, 2013). Since the target of ibrutinib (BTK) is a known downstream target of CXCR4, we sought to clarify if ibrutinib activity in WM LPCs was modulated by WHIM-like mutations in CXCR4. Methods We first sought to confirm the frequency of WHIM-like mutations in 87 untreated WM patients by Sanger sequencing. The most common CXCR4 somatic mutation identified (S338X) in these studies was then cloned by PCR from CD19+ LPCs from a WM patient with this somatic mutation. Wild type (WT) and S338X CXCR4 cDNAs were subcloned into plenti-IRES-GFP vector, and transduced using an optimized lentiviral based strategy into BCWM.1 WM cells. Five days after transduction, GFP positive cells were sorted and used for functional studies. Surface expression of CXCR4 was determined by flow cytometeric analysis using a PE-conjugated anti-CXCR4 monoclonal antibody. The expression of phosphorylated BTK, AKT, and ERK1/2 was determined by western blot analysis. Cell proliferation was measured with alamar blue. Results Sanger sequencing identified nonsense or frame shift mutations (WHIM-like) in the c-terminal tail of CXCR4 in 28 of 87 (32%) patients, the most common of which was a non-sense mutation (S338X) that was present in 12 patients. BCWM.1 cells were then transduced with control vector, CXCR4 wild type or CXCR4 S338X mutant expressing vectors. Expression was confirmed by cDNA Sanger sequencing. Stably transduced cells exposed to ibrutinib (0.5uM or 1uM) showed significantly reduced cell proliferation (p<0.005). Ibrutinib treated control vector and CXCR4 wild-type transduced cells showed suppressed tumor cell growth even in the presence of the CXCR4 ligand CXCL12 (20 nM), whereas cells transduced with CXCR4 S338X WHIM-like mutation demonstrated resistance to ibrutinib growth effect (p<0.005). In turn, this rescue could be blocked by treatment with 30uM of the CXCR4 specific inhibitor AMD3100 confirming that this effect was mediated through CXCR4 (p<0.005) (Figure 1). Phosphorylated BTK, ERK1/2 and AKT signaling increased following CXCL12 stimulation in all transduced cells, while ibrutinib inhibited their activation in control vector and CXCR4 wild-type, but not CXCR4 S338X mutant cells. CXCR4 triggered signaling by CXCL12 in these experiments was confirmed by pre-treatment with AMD3100. Conclusions By Sanger sequencing, WHIM-like CXCR4 somatic mutations are observed in 1/3 of untreated WM patients. WHIM-like CXCR4 mutations are associated with resistance to ibrutinib mediated ERK1/2 and AKT signaling, as well as growth suppression in the presence of the CXCR4 ligand, CXCL12, in WM cells. These studies may have important implications for CXCR4 modulation in the treatment of WM, as well as potential use of CXCR4 mutations in predicting outcome for patients undergoing ibrutinib therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals ecTMB: a robust method to estimate and classify tumor mutational burden

2019 ◽  
Author(s):  
Lijing Yao ◽  
Yao Fu ◽  
Marghoob Mohiyuddin ◽  
Hugo YK Lam
Keyword(s):  
Somatic Mutations ◽  
Clinical Decision Making ◽  
Patient Survival ◽  
Clinical Decision ◽  
Statistical Framework ◽  
Mutational Burden ◽  
Mutation Model ◽  
Tumor Mutational Burden ◽  
Multiple Challenges

AbstractTumor Mutational Burden (TMB) is a measure of the abundance of somatic mutations in a tumor, which has been shown to be an emerging biomarker for both anti-PD-(L)1 treatment and prognosis. Nevertheless, multiple challenges still hinder the adoption of TMB for clinical decision-making. The key challenges are the inconsistency of TMB measurement among assays and a lack of meaningful threshold for TMB classification. We describe a powerful and flexible statistical framework for estimation and classification of TMB (ecTMB). ecTMB uses an explicit background mutation model to predict TMB robustly and consistently. In addition to the two known TMB subtypes, TMB-high and TMB-low, we discovered a novel TMB subtype, named TMB-extreme, which was significantly associated with patient survival outcome. This discovery enabled ecTMB to classify samples to biologically and clinically relevant subtypes defined by TMB.

Exome Sequencing Identifies Somatic Mutations of DDX3X in Natural Killer/T-Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1663-1663
Author(s):  
Lu Jiang ◽  
Zhao-Hui Gu ◽  
Zi-Xun Yan ◽  
Xia Zhao ◽  
Yin-Yin Xie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
T Cell ◽  
Natural Killer ◽  
Expression Profiling ◽  
Somatic Mutations ◽  
Cell Lymphoma ◽  
Rna Helicase ◽  
Natural Killer T Cell ◽  
Killer T Cell

Abstract Natural-killer/T cell lymphoma (NKTCL) is a malignant proliferation of CD56+/cytoCD3+ lymphocytes and constitutes a heterogeneous group of aggressive lymphoma prevalent in Asian and South American populations. NKTCL represents a distinct clinicopathologic entity of non-Hodgkin’s lymphoma, characterized by male predominance, strong association with Epstein-Barr virus (EBV) infection, prominent tissue necrosis and aggressive clinical course. However, molecular pathogenesis of NKTCL remains largely elusive. Here we identified somatic mutations by whole-exome sequencing in 25 NKTCL patients and extended validation through targeted sequencing in an additional 80 cases. Functional experiments including RNA unwinding test, colony forming assay, cell proliferation assay and gene expression profiling were also performed. Overall, 50.5% of NKTCL patients displayed somatic mutations of RNA helicase family, tumor suppressors (TP53 and MGA), and/or epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3). Recurrent mutations were most frequently discovered in RNA helicase gene DDX3X (21/105 cases, 20.0%). Mutations of DDX3X were seldom overlapped with those of TP53. Functionally, DDX3X mutants exhibited reduced RNA unwinding activity and enhanced cell proliferation. Similar stimulatory effect on cell proliferation was observed in cells transfected with specific siRNA targeting DDX3X. Gene expression profiling revealed an association of DDX3X mutations with activation of NF-kB and MAPK pathways. The clinical follow-up data showed that DDX3X-mutated patients presented a poor prognosis. Our work suggests the heterogeneity of gene mutational spectrum of NKTCL and provides a potential therapeutic target for relevant cases. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4022
Author(s):  
Donatella Treppiedi ◽  
Anna Maria Barbieri ◽  
Genesio Di Muro ◽  
Giusy Marra ◽  
Federica Mangili ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Binding ◽  
Somatic Mutations ◽  
Proteolytic Cleavage ◽  
Proximity Ligation Assay ◽  
Binding Motif ◽  
Transfected Cells ◽  
Significant Difference ◽  
Acth Secreting

Cushing’s Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in an excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases USP8 and USP48, and in BRAF genes, have been reported in a subset of patients affected by CD. The aim of this study was to characterize the genetic profile of a cohort of 60 patients with ACTH-secreting tumors, searching for somatic mutations in USP8, USP48, and BRAF hotspot regions. Seven patients were found to carry USP8 somatic mutations in the well-characterized 14-3-3 protein binding motif (n = 5 P720R, n = 1 P720Q, n = 1 S718del); 2 patients were mutated in USP48 (M415I); no mutation was identified in BRAF. In addition, a novel USP8 variant, G664R, located in exon 14, upstream of the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of USP8 G664R variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the USP8 G664R variant resulted in a significant increase of ACTH release and cell proliferation (+114.5 ± 53.6% and +28.3 ± 2.6% vs. empty vector transfected cells, p < 0.05, respectively). Notably, USP8 proteolytic cleavage was enhanced in AtT-20 cells transfected with G664R USP8 (1.86 ± 0.58–fold increase of N-terminal USP8 fragment, vs. WT USP8, p < 0.05). Surprisingly, in situ Proximity Ligation Assay (PLA) experiments showed a significant reduction of PLA positive spots, indicating USP8/14-3-3 proteins colocalization, in G664R USP8 transfected cells with respect to WT USP8 transfected cells (−47.9 ± 6.6%, vs. WT USP8, p < 0.001). No significant difference in terms of ACTH secretion, cell proliferation and USP8 proteolytic cleavage, and 14-3-3 proteins interaction was observed between G664R USP8 and S718del USP8 transfected cells. Immunofluorescence experiments showed that, contrary to S718del USP8 but similarly to WT USP8 and other USP8 mutants, G664R USP8 displays an exclusive cytoplasmic localization. In conclusion, somatic mutations were found in USP8 (13.3% vs. 36.5% incidence of all published mutations) and USP48 (3.3% vs. 13.3% incidence) hotspot regions. A novel USP8 variant was identified in a CD patient, and in vitro functional studies in AtT-20 cells suggested that this somatic variant might be clinically relevant in ACTH-secreting tumor pathogenesis, expanding the characterization of USP8 functional domains.

scholarly journals The Hippo pathway origin and its oncogenic alteration in evolution

2019 ◽  
Author(s):  
Yuxuan Chen ◽  
Han Han ◽  
Gayoung Seo ◽  
Rebecca Vargas ◽  
Bing Yang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Evolutionary History ◽  
Somatic Mutations ◽  
Hippo Pathway ◽  
Evolutionary Perspective ◽  
Paralogous Gene ◽  
Evolutionarily Conserved ◽  
History Of ◽  
Unicellular Origin ◽  
The Hippo Pathway

AbstractThe Hippo pathway is a central regulator of organ size and a key tumor suppressor via coordinating cell proliferation and death. Initially discovered in Drosophila, the Hippo pathway has been implicated as an evolutionarily conserved pathway in mammals; however, how this pathway was evolved to be functional from its origin is still largely unknown. In this study, we traced the Hippo pathway in premetazoan species, characterized the intrinsic functions of its ancestor components, and unveiled the evolutionary history of this key signaling pathway from its unicellular origin. In addition, we elucidated the paralogous gene history for the mammalian Hippo pathway components and characterized their cancer-derived somatic mutations from an evolutionary perspective. Taken together, our findings not only traced the conserved function of the Hippo pathway to its unicellular ancestor components, but also provided novel evolutionary insights into the Hippo pathway organization and oncogenic alteration.

scholarly journals Exome sequencing reveals mutant genes with low penetrance involved in MEN2A-associated tumorigenesis

2014 ◽  
Vol 22 (1) ◽  
pp. 23-33 ◽  
Author(s):  
Jie Cai ◽  
Lin Li ◽  
Lei Ye ◽  
Xiaohua Jiang ◽  
Liyun Shen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Exome Sequencing ◽  
Somatic Mutations ◽  
Snp Array ◽  
Point Mutations ◽  
Causative Mutation ◽  
Endocrine Tumors ◽  
Mapk Phosphorylation ◽  
Recurrent Mutations ◽  
Associated Tumors

Activating rearranged during transfection (RET) mutations function as the initiating causative mutation for multiple endocrine neoplasia type 2A (MEN2A). However, no conclusive findings regarding the non-RET genetic events have been reported. This is the first study, to our knowledge, examining genomic alterations in matched MEN2A-associated tumors. We performed exome sequencing and SNP array analysis of matched MEN2A tumors and germline DNA. Somatic alterations were validated in an independent set of patients using Sanger sequencing. Genes of functional interest were further evaluated. The germline RET mutation was found in all MEN2A-component tumors. Thirty-two somatic mutations were identified in the nine MEN2A-associated tumors, of which 28 (87.5%) were point mutations and 4 (12.5%) were small insertions, duplications, or deletions. We sequenced all the mutations as well as coding sequence regions of the 12 genes in an independent sample set including 35 medullary thyroid cancers (20 MEN2A) and 34 PCCs (22 MEN2A), but found no recurrent mutations. Recurrent alterations were found in 13 genes with either mutations or alterations in copy number, including an EIF4G1 mutation (p. E1147V). Mutation of EIF4G1 led to increased cell proliferation and RET/MAPK phosphorylation, while knockdown of EIF4G1 led to reduced cell proliferation and RET/MAPK phosphorylation in TT, MZ-CRC1, and PC-12 cells. We found fewer somatic mutations in endocrine tumors compared with non-endocrine tumors. RET was the primary driver in MEN2A-associated tumors. However, low-frequency alterations such as EIF4G1 might participate in MEN2A-associated tumorigenesis, possibly by regulating the activity of the RET pathway.

Fibroblasts During Hypertrophic Scar Formation and Resolution

1973 ◽  
Vol 31 ◽  
pp. 428-429
Author(s):  
C. W. Kischer
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Cell Proliferation ◽  
Fine Structure ◽  
Metabolic Activity ◽  
Hypertrophic Scar ◽  
Normal Skin ◽  
Ground Substance ◽  
Hypertrophic Scars ◽  
Scanning Electron

The morphology of the fibroblasts changes markedly as the healing period from burn wounds progresses, through development of the hypertrophic scar, to resolution of the scar by a self-limiting process of maturation or therapeutic resolution. In addition, hypertrophic scars contain an increased cell proliferation largely made up of fibroblasts. This tremendous population of fibroblasts seems congruous with the abundance of collagen and ground substance. The fine structure of these cells should reflect some aspects of the metabolic activity necessary for production of the scar, and might presage the stage of maturation.A comparison of the fine structure of the fibroblasts from normal skin, different scar types, and granulation tissue has been made by transmission (TEM) and scanning electron microscopy (SEM).

The Effect of Androgen Depletion and Replacement on the Epithelium of the Seminal Vesicle of the Aging Rat

1975 ◽  
Vol 33 ◽  
pp. 590-591
Author(s):  
Venita F. Allison
Keyword(s):  
Cell Proliferation ◽  
Seminal Vesicle ◽  
Male Rats ◽  
Target Cells ◽  
Cell Regeneration ◽  
Secretory Function ◽  
Electron Microscopic ◽  
Aging Rat ◽  
Microscopic Studies ◽  
Androgen Depletion

In 1930, Moore, Hughes and Gallager reported that after castration seminal vesicle epithelial cell atrophy occurred and that cell regeneration could be achieved with daily injections of testis extract. Electron microscopic studies have confirmed those observations and have shown that testosterone injections restore the epithelium of the seminal vesicle in adult castrated male rats. Studies concerned with the metabolism of androgens point out that dihydrotestosterone stimulates cell proliferation and that other metabolites of testosterone probably influence secretory function in certain target cells.Although the influence of androgens on adult seminal vesicle epithelial cytology is well documented, little is known of the effect of androgen depletion and replacement on those cells in aging animals. The present study is concerned with the effect of castration and testosterone injection on the epithelium of the seminal vesicle of aging rats.

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