Choice of control tissue impacts designation of germline variants in a cohort of papillary thyroid carcinoma patients.

BACKGROUND: The term germline is commonly used to refer to any non-tumor control sample analyzed in tumor-normal paired sequencing experiments. Blood is the most commonly utilized control, and variants found in both tumor and blood are considered germline. However, somatic variants accumulate within an organism from embryogenesis throughout life. The resultant mosaicism is extensive and calls into question the assumption that blood, or any somatic tissue, represents the germline. Misclassification of germline and somatic variants has critical consequences for individual patient care and enormous impact on our health care system, given potential screening, counseling, and treatment implications of misidentifying germline variants. PATIENTS AND METHODS: Whole-exome sequencing was performed on six separate specimens from each of two patients with papillary thyroid carcinoma, and three specimens each from eight additional patients forming a validation cohort. Tumor variants were compared with each individual non-tumor control and with composite control sets generated as approximations of true germline. For the index patient, parental blood was also sequenced to assess whether patient-only samples could approximate a trio-derived germline. RESULTS: Using different non-tumor control tissues results in altered germline-somatic designation of tumor variants. In patient 1, 82% of variants are labeled germline using blood control, compared with 75.8%, 61.5%, and 49.6% using lymph node, thyroid, and thymus, respectively. In patient 2, the thyroid control resulted in the greatest percentage of germline calls (70.0%), followed by thymus (56.0%), lymph node (50.1%), and blood (44.1%). Composite control sets built from multiple samples can approximate the germline, even in the absence of parental DNA. CONCLUSIONS: Misclassification of germline-somatic origin has potential consequences for patient care, informing screening, trial eligibility, prophylactic interventions, and family planning. This study demonstrates the need for caution in interpreting germline-somatic designation if these data are to inform clinical decisions and suggests that improved design of controls can overcome current limitations.

1,25-dihydroxyvitamin D3 and retonic acid analogues induce differentiation in breast cancer cells with function- and cell-specific additive effects.

Vitamin D3 derivatives and retinoids can induce cell cycle arrest, differentiation and cell death in many cell lines. These compounds can act cooperatively in some of their functions and may be of potential use either individually or in combination in the treatment of breast cancer. The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), all-trans retinoic acid (ATRA) and several analogues were evaluated on malignant phenotypic traits of breast cancer cell lines MCF-7, T-47D and MDA-MB-231. Both 1,25(OH)2D3 and ATRA caused a decrease in anchorage independent colony formation in MCF-7 and T-47D cells in a dose-dependent manner. The effects of 1,25(OH)2D3 10(-10) and 10(-9) M were synergistic with ATRA 10(-8) M in T-47D cells but were antagonistic in both MCF-7 and in T-47D cells at most concentrations. Both 1,25(OH)2D3 and ATRA individually induced an accumulation of MCF-7 cells in the G1 phase of the cell cycle and an associated increase in p21WAFI/CiP1, p27KiP1 and a dephosphorylation of Rb but the effects were not additive. Both compounds inhibited the invasive capacity of MDA-MB-231 cells. 1,25(OH)2D3 but not ATRA caused an increase in E-cadherin levels in MDA-MB-231 cells. These two functions were not additive. The compounds 1,25(OH)2D3, a noncalcemic analogue 1,25(OH)2-16-ene-23-yne-D3, ATRA, AGN195183, an RARalpha-specific agonist, and AGN190168 (tazarotene), an RARbeta/gamma-selective agonist, induced differentiation as determined by measurements of lipid droplet formation. The individual effects of 1,25(OH)2-16-ene-23-yne-D3 combined with ATRA or with tazarotene at 10(-9) M each were additive in MCF-7 and MDA-MB-231 cells on lipid formation. The data demonstrate that both 1,25(OH)2D3, ATRA, and selected analogues induce a more differentiated phenotype in breast cancer cells with additive effects that are function- and cell-specific.

Intracellular FGF-2 promotes differentiation in T-47D breast cancer cells.

To test the implicated role of basic fibroblast growth factor (bFGF; FGF-2) in promoting differentiation in breast cancer, we enforced the expression of FGF-2 in T-47D breast cancer cells. Expression of FGF-2 conferred an overall less malignant phenotype to T-47D cells as revealed by their reduced proliferative response, impaired capacity for anchorage-independent growth, and invasion through Matrigel. To understand one candidate mechanism for the intracellular FGF-2-mediated anti-invasive effect, we examined the effect of FGF-2 on T-47D cell motility. Addition of recombinant FGF-2 to the growth medium markedly enhanced cell motility while constitutive expression of intracellular FGF-2 significantly inhibited the migratory potential of T-47D cells in a dominant manner. FGF-2-expressing T-47D cells also formed relatively defined branching structures in Matrigel matrices, a characteristic phenotype of differentiation in breast cancer cells. These data suggest a potential role for FGF-2 in promoting functional differentiation of breast epithelial cells.

In vivo disruption of the fas pathway abrogates gastric growth alterations secondary to Helicobacter infection.

Helicobacter infection is associated with gastric cell growth alterations, plausibly predisposing to ulcer disease and gastric adenocarcinoma. Previous investigations from our laboratory have implicated the involvement of the Fas pathway in Helicobacter-induced apoptotic signaling in vitro. In this report we use C57BL/6J00064 mice to examine the direct role of Fas signaling in Helicobacter-mediated growth alterations in vivo. Helicobacter infection up-regulated gastric cell Fas antigen (Fas Ag) mRNA and increased surface receptor expression, along with concomitant altered apoptotic and proliferative response, measured by terminal deoxytransferase-deoxyuridine 5'-triphosphate nick end labeling and 5-bromo-2'-deoxuridine immunohistochemistry, respectively. In addition, histopathological alterations, including parietal cell loss and gastric atrophy, were noted. In contrast, infection in B6. MRL-FAS(lpr), a Fas Ag knockout mouse in the C57BL/6 background, did not result in increased apoptosis, proliferation, or histological alterations, a finding that argues strongly for the role of Fas-signaling pathway in orchestrating diverse growth responses to Helicobacter infection.

Basic fibroblast growth factor confers a less malignant phenotype in MDA-MB-231 human breast cancer cells.

Basic fibroblast growth factor (FGF-2) expression is associated with a more differentiated phenotype, earlier stage of disease, and a better prognosis in breast cancer patients. To determine whether expression of FGF-2 can cause a less malignant phenotype, we engineered MDA-MB-231 cells, a highly dedifferentiated, invasive breast cancer cell line, to express different isoforms of FGF-2. Cells expressed either cytoplasmic, nuclear, or a combination of both FGF-2 isoforms. Western blots of 2 M NaCl washes and of conditioned medium demonstrated that these cells did not export FGF-2. Cells expressing FGF-2 had levels of fibroblast growth factor receptors equivalent with those of control cells. Transformation was assayed by anchorage-independent colony formation and tumor formation in athymic mice. All of the constructs expressing various FGF-2 isoforms had a 60-70% reduction in colony formation in soft agar, but only cells expressing the Mr 18,000 FGF-2 isoform formed fewer and smaller tumors in mice. To determine potential mechanisms responsible for a less malignant phenotype, experiments measuring invasion in Matrigel, the secretion of matrix metalloprotease activity and migration in a modified Boyden chamber and in a patch wound motility assay were carried out. Cells expressing the Mr 18,000 cytoplasmic FGF-2 moiety had a 45% decrease in invasion in Matrigel compared to vector-transfected controls. Cells expressing Mr 18,000 FGF-2 had an increase in Mr 97,000 and Mr 48,000 collagenase, demonstrating that the decreased invasive potential was not due to a down-regulation of gelatinolytic or caseinolytic matrix metalloproteinases. However, motility was decreased in both assays, primarily in cells expressing Mr 18,000 FGF-2, whereas exogenous recombinant human FGF-2 had no effect. These studies demonstrate for the first time that FGF-2 expression can cause a less malignant phenotype in breast cancer cells, possibly as a result of decreased motility and invasion.

Tumor necrosis factor alpha and interleukin 1beta up-regulate gastric mucosal Fas antigen expression in Helicobacter pylori infection.

Fas-mediated gastric mucosal apoptosis is gaining attention as a cause of tissue damage due to Helicobacter pylori infection. We explored the effects of H. pylori directly, and the effects of the inflammatory environment established subsequent to H. pylori infection, on Fas-mediated apoptosis in a nontransformed gastric mucosal cell line (RGM-1). Exposure to H. pylori-activated peripheral blood mononuclear cells (PBMCs), but not H. pylori itself, induced Fas antigen (Fas Ag) expression, indicating a Fas-regulatory role for inflammatory cytokines in this system. Of various inflammatory cytokines tested, only interleukin 1beta and tumor necrosis factor alpha induced Fas Ag expression, and removal of either of these from the conditioned medium abrogated the response. When exposed to Fas ligand, RGM-1 cells treated with PBMC-conditioned medium underwent massive and rapid cell death, interestingly, with a minimal effect on total cell numbers early on. Cell cycle analysis revealed a substantial increase in S phase cells among cells exposed to Fas ligand, suggesting an increase in their proliferative response. Taken together, these data indicate that the immune environment secondary to H. pylori infection plays a critical role in priming gastric mucosal cells to undergo apoptosis or to proliferate based upon their Fas Ag status.

Fas receptor up-regulation and membrane localization concurrent with apoptosis in idiopathic esophageal ulceration: toward a better understanding of esophageal injury in AIDS.

Activation of the Fas-mediated apoptotic pathway in the etiology of idiopathic esophageal ulcerations (IEUs) was investigated. Constitutive expression of Fas ligand (Fas L) was found in the basal layer of the normal esophageal mucosa and in IEUs and cytomegalovirus ulcerations. In addition to an altered cytokine environment, there was significant up-regulation of Fas antigen mRNA and membrane localization of the receptor in IEU specimens. Concomitant increase in the basic components of Fas machinery, together with elevated mucosal apoptosis, strongly suggests the potential role of the Fas signaling pathway in the onset of tissue destruction associated with IEUs.

Apoptosis in Helicobacter pylori-associated gastric and duodenal ulcer disease is mediated via the Fas antigen pathway.

Increased mucosal apoptosis is seen in H. pylori-infected gastric tissue; however, the precise mechanism by which this organism triggers programmed cell death is poorly understood and is investigated in this study. One pathway for induction of apoptosis is the Fas Ag pathway. Normal gastric and small bowel tissue express low levels of Fas antigen and nondetectable levels of Fas ligand. Consequent to H. pylori infection, there is elevated expression of Fas antigen in mucosal cells concurrent with Fas ligand expressing lymphocytes. This prompted us to investigate the potential role of Fas in mediating H. pylori-related apoptosis. It has been shown that inflammatory cytokines are abundant in H. pylori-infected tissue and that cytokines regulate the expression of Fas Ag in various tissue types. Using cell culture, we examine the role of specific inflammatory cytokines in activating this pathway. This communication presents the first evidence to implicate the Fas pathway in mediating apoptosis in H. pylori-associated gastric and duodenal ulcer disease.

A role for apoptosis in the pathogenesis of AIDS-related idiopathic esophageal ulcers.

Lack of understanding of the mechanism of tissue destruction associated with idiopathic esophageal ulcers (IEUs) poses a diagnostic and therapeutic dilemma for the clinician. The possible role of apoptosis in IEUs, as suggested by endoscopic and histologic observations, was investigated by examination of archival tissues for apoptosis-related DNA fragmentation using in situ nick end labeling (TUNEL). High levels of apoptosis were observed in mucosal cells immediately adjacent to IEUs. Apoptotic cells were virtually absent in normal control tissues, while the edges and bases of lesions and sloughed-off tissues in IEUs in human immunodeficiency virus (HIV)-infected patients showed elevated levels of apoptotic cell death. However, tissue samples from patients with esophageal ulcerations of known etiology showed no apoptosis of mucosal cells. These data support a role for apoptosis in the pathogenesis of IEUs and suggest a mechanism involving HIV-associated bystander killing of uninfected mucosal cells.

Characterization of c-myc-transformed rat fibroblasts resistant to apoptosis induced ny growth factor deprivation.

Under appropriate conditions (e.g., growth factor withdrawal), the deregulated expression of c-myc in rodent fibroblasts leads to substantial cell death due to apoptosis. To better understand this process, we selected for c-myc-transformed Rat1A fibroblasts that were resistant to growth factor deprivation-induced cell death. One clonal isolate exhibited prolonged survival in serum-free medium and displayed reduced levels of apoptosis-related DNA fragmentation. These cells were also resistant to induction of apoptosis by the protein kinase inhibitor staurosporine. They retained a transformed cell phenotype and expressed the proviral human c-myc allele in an unaltered fashion, strongly indicating that the mutation of a cellular gene other than c-myc accounts for the apoptosis-resistant phenotype. The results of somatic cell hybrid analysis of this cell line are consistent with a recessive mutation. Our findings suggest a novel mechanism for abrogation of apoptosis in neoplastic cells and provide a model system for the study of its role in tumorigenesis and resistance to antineoplastic therapy.

Mutagenic and recombinagenic effects of diethylstilbestrol quinone.

Estrogens are believed to be major contributors to many cancers of the human female genital tract, but the mechanism of their carcinogenic action is not well-understood. While a tumor-promoting role for estrogens is well-supported, whether they also act as tumor initiators has remained controversial. Here, we have sought to examine the mutagenic potential of diethylstilbestrol, a synthetic estrogen that is a powerful carcinogen in hamsters, and is suspected to be a human carcinogen. Phage M13 single-stranded DNA was treated in vitro with diethylstilbestrol quinone (DES Q: 1.25 mM) and transfected into Escherichia coli cells. DES Q treatment resulted in an apparent enhancement of mutagenesis in the LacZ(alpha) gene segment. DNA sequence analysis of LacZ(alpha) mutants obtained by transfection of DES Q-treated DNA revealed that the major effect of DES Q treatment has been a 6-fold elevation of recombination between the phage-borne LacZ(alpha) sequence and the LacZ delta M15 sequence on the E. coli fertility plasmid F. To confirm whether DES Q treatment is recombinagenic, we used an experimental system that allows the detection of recombination between a defective E. coli chromosomal LacY gene and a normal counterpart borne on a plasmid. Transfection of DES Q (0.06-12 mM) treated plasmid DNA showed significant enhancement (2-100-fold) in recombination, but not in mutagenesis. These results raise the possibility that estrogen quinones may induce recombinagenic DNA damage.