Three-dimensional nuclear architecture distinguishes thyroid cancer histotypes.

Molecular markers can serve as diagnostic tools to support pathological analysis in thyroid neoplasms. However, because the same markers can be observed in some benign thyroid lesions, additional approaches are necessary to differentiate thyroid tumor subtypes, prevent overtreatment and tailor specific clinical management. This applies particularly to the recently described variant of thyroid cancer referred to as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). This variant has an estimated prevalence of 4.4% to 9.1% of all papillary thyroid carcinomas worldwide. We studied 60 thyroid lesions: 20 classical papillary thyroid carcinoma (CPTC), 20 follicular variant of PTC (FVPTC) and 20 NIFTP. We examined morphological and molecular features to identify parameters that can differentiate NIFTP from the other PTC subtypes. When blindly investigating the nuclear architecture of thyroid neoplasms, we observed that NIFTP has significantly longer telomeres than CPTC and FVPTC. Super-resolved 3D-structured illumination microscopy demonstrated that NIFTP is heterogeneous and that its nuclei contain more densely packed DNA and smaller interchromatin spaces than CPTC and FVPTC, a pattern that resembles normal thyroid tissue. These data are consistent with the observed indolent biological behavior and favorable prognosis associated with NIFTP, which lacks BRAFV600E mutations. Of note, next-generation thyroid oncopanel sequencing was unable to distinguish the thyroid cancer histotypes in our study cohort. In summary, our data suggest that 3D nuclear architecture can be a powerful analytical tool to diagnose and guide clinical management of NIFTP.

Three-dimensional telomere profiles in papillary thyroid cancer variants: A pilot study.

Besides the two main histologic types of papillary thyroid carcinoma (PTC), the classical PTC (CL-PTC) and the follicular variant PTC (FV-PTC), several other variants are described. The encapsulated FV-PTC variant was recently reclassified as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) due to its similarities to benign lesions. Specific molecular signatures, however, are still unavailable. It is well known that improper DNA repair of dysfunctional telomeres may cause telomere-related genome instability. The mechanisms involved in the damaged telomere repair processing may lead to detrimental outcomes, altering the three-dimensional (3D) nuclear telomere and genome organization in cancer cells. This pilot study aimed to evaluate whether a specific 3D nuclear telomere architecture might characterize NIFTP, potentially distinguishing it from other PTC histologic variants. Our findings demonstrate that 3D telomere profiles of CL-PTC and FV-PTC were different from NIFTP and that NIFTP more closely resembles follicular thyroid adenoma (FTA). NIFTP has longer telomeres than CL-PTC and FV-PTC samples, and the telomere length of NIFTP overlaps with that of the FTA histotype. In contrast, there was no association between BRAF expression and telomere length in all tested samples. These preliminary findings reinforce the view that NIFTP is closer to non-malignant thyroid nodules and confirm that PTC features short telomeres.

Genomic Instability in Circulating Tumor Cells.

Circulating tumor cells (CTCs) can promote distant metastases and can be obtained through minimally invasive liquid biopsy for clinical assessment in cancer patients. Having both genomic heterogeneity and instability as common features, the genetic characterization of CTCs can serve as a powerful tool for a better understanding of the molecular changes occurring at tumor initiation and during tumor progression/metastasis. In this review, we will highlight recent advances in the detection and quantification of tumor cell heterogeneity and genomic instability in CTCs. We will focus on the contribution of chromosome instability studies to genetic heterogeneity in CTCs at the single-CTC level by discussing data from different cancer subtypes and their impact on diagnosis and precision medicine.

It is ethical to diagnose a public figure one has not personally examined.

Should psychiatrists be able to speculate in the press or social media about their theories? John Gartner argues the risk to warn the public of concerns about public figures overrides the duty of confidentiality; whereas Alex Langford suggests this is beyond the ethical remit of psychiatric practice.Declaration of interestA.O'B is joint debates and analysis Editor of the British Journal of Psychiatry. J.G. is the founder of Duty To Warn, an association of mental health professionals who advocate the president's removal under the 25th Amendment on the grounds that he is psychologically unfit and dangerous.

Distinct 3D Structural Patterns of Lamin A/C Expression in Hodgkin and Reed-Sternberg Cells.

Classical Hodgkin's lymphoma (cHL) is a B-Cell lymphoma comprised of mononuclear Hodgkin cells (H) and bi- to multi-nucleated Reed-Sternberg (RS) cells. Previous studies revealed that H and RS cells express lamin A/C, a component of the lamina of the nuclear matrix. Since no information was available about the three-dimensional (3D) expression patterns of lamin A/C in H and RS cells, we analyzed the 3D spatial organization of lamin in such cells, using 3D fluorescent microscopy. H and RS cells from cHL derived cell lines stained positive for lamin A/C, in contrast to peripheral blood lymphocytes (PBLs), in which the lamin A/C protein was not detected or weak, although its presence could be transiently increased with lymphocyte activation by lipopolysaccharide (LPS). Most importantly, in H and RS cells, the regular homogeneous and spherically shaped lamin A/C pattern, identified in activated lymphocytes, was absent. Instead, in H and RS cells, lamin staining showed internal lamin A/C structures, subdividing the nuclei into two or more smaller compartments. Analysis of pre-treatment cHL patients' samples replicated the lamin patterns identified in cHL cell lines. We conclude that the investigation of lamin A/C protein could be a useful tool for understanding nuclear remodeling in cHL.

Identification of Neuroblastoma Subgroups Based on Three-Dimensional Telomere Organization.

Using 3D telomere quantitative fluorescence in situ hybridization, we determined the 3D telomere organization of 74 neuroblastoma tissue samples. Hierarchical cluster analysis of the measured telomere parameters identified three subgroups from our patient cohort. These subgroups have unique telomere profiles based on telomere length and nuclear architecture. Subgroups with higher levels of telomere dysfunction were comprised of tumors with greater numbers of telomeres, telomeric aggregates, and short telomeres (P<.0001). Tumors with greater telomere dysfunction were associated with unfavorable tumor characteristics (greater age at diagnosis, unfavorable histology, higher stage of disease, MYCN amplification, and higher MYCN expression) and poor prognostic risk (P<.001). Subgroups with greater telomere dysfunction also had higher intratumor heterogeneity. MYCN overexpression in two neuroblastoma cell lines with constitutively low MYCN expression induced changes in their telomere profile that were consistent with increased telomere dysfunction; this illustrates a functional relationship between MYCN and 3D telomere organization. This study demonstrates the ability to classify neuroblastomas based on the level of telomere dysfunction, which is a novel approach for this cancer.

MYCN overexpression is associated with unbalanced copy number gain, altered nuclear location, and overexpression of chromosome arm 17q genes in neuroblastoma tumors and cell lines.

MYCN amplification and MYCN overexpression are poor prognostic factors in neuroblastoma. Tumors with unbalanced chromosome arm 17q gain are often associated with MYCN amplification; however, the relationship between chromosome 17 copy number status and MYCN expression is not known. We investigated the relationship between MYCN expression and chromosome 17 copy number, nuclear location, and gene expression. By performing dual-colored fluorescence in situ hybridization on 16 primary neuroblastomas, we found that those with unbalanced gain of 17q have high MYCN expression, those with no gain have medium expression, and those with numerical gain have low expression (P < 0.0001). We also found that the nuclear location of 17q correlates with chromosome 17 copy number status: copies in tumors with unbalanced gain and no gain of chromosome 17 occupy a more central location than those in tumors with balanced gain (P < 0.0001). We show that a more central nuclear location of 17q coincides with increased expression of genes found within this chromosome arm. To further understand the association between MYCN expression and chromosome 17, we overexpressed MYCN in two low-expressing MYCN cell lines, SHEP and GIMEN. We found that both cell lines had an unbalanced gain of chromosome 17q, a more central nuclear location of the region and increased expression of the 17q genes. Therefore, this study indicates, for the first time, a functional relationship between MYCN overexpression and the gain of 17q in neuroblastoma.

Surficial redistribution of fallout ¹³¹iodine in a small temperate catchment.

Isotopes of iodine play significant environmental roles, including a limiting micronutrient ((127)I), an acute radiotoxin ((131)I), and a geochemical tracer ((129)I). But the cycling of iodine through terrestrial ecosystems is poorly understood, due to its complex environmental chemistry and low natural abundance. To better understand iodine transport and fate in a terrestrial ecosystem, we traced fallout (131)iodine throughout a small temperate catchment following contamination by the 11 March 2011 failure of the Fukushima Daiichi nuclear power facility. We find that radioiodine fallout is actively and efficiently scavenged by the soil system, where it is continuously focused to surface soils over a period of weeks following deposition. Mobilization of historic (pre-Fukushima) (137)cesium observed concurrently in these soils suggests that the focusing of iodine to surface soils may be biologically mediated. Atmospherically deposited iodine is subsequently redistributed from the soil system via fluvial processes in a manner analogous to that of the particle-reactive tracer (7)beryllium, a consequence of the radionuclides' shared sorption affinity for fine, particulate organic matter. These processes of surficial redistribution create iodine hotspots in the terrestrial environment where fine, particulate organic matter accumulates, and in this manner regulate the delivery of iodine nutrients and toxins alike from small catchments to larger river systems, lakes and estuaries.

Role of thymic stromal lymphopoietin (TSLP) in palifermin-mediated immune modulation and protection from acute murine graft-versus-host disease.

Using the C57BL/6→(C57BL/6 x DBA/2)F(1)-hybrid model of acute graft-versus-host disease (GVHD), we previously showed that treating the donor mice with palifermin provides protection from morbidity and a shift from Th1 to Th2 cytokine production. To determine whether thymic stromal lymphopoietin (TSLP) is involved in palifermin-mediated immune modulation, we used donors from the following groups: (1) untreated wild-type donors, (2) palifermin-treated wild-type donors, (3) untreated TSLPR(-/-) donors, and (4) palifermin-treated TSLPR(-/-) donors. Survival in the recipients was 0%, 100%, 31%, and 0%, for groups 1-4, respectively, indicating that TSLP responsiveness is required for palifermin-mediated protection from GVHD. We also found that the increases in Th2 cytokine levels that are induced by palifermin treatment are obviated in TSLPR(-/-) donors, and that protection from GVHD (group 2) is associated with a higher percentage of CD4(+)CD25(+)Foxp3(+) cells in the graft. Collectively, our findings show that when palifermin and TSLP act in concert, the predominant effect is protection in this model.

Palifermin mediates immunoregulatory effects in addition to its cytoprotective effects in mice with acute graft-versus-host disease.

INTRODUCTION: Treating recipient mice with palifermin (recombinant human keratinocyte growth factor) prevents the development of acute, lethal, graft-versus-host disease (GVHD). This is due, at least in part, to the ability of palifermin to protect epithelial cells from injury. Using the C57BL/6-->(C57BL/6 x DBA/2)F(1)-hybrid model, we previously showed that the protective effect of palifermin was also associated with redirection of the cytokine profile from Th1 to Th2. DISCUSSION: To study this immunoregulatory effect more directly, we induced acute GVH reactions in which we treated the donors rather than the recipients with palifermin. The recipient mice were protected from GVHD-associated morbidity, and their cytokine profile was predominantly Th2. The palifermin-treated donor mice alone showed a similar Th2 cytokine profile, and we observed elevated levels of thymic stromal lymphopoietin mRNA in the thymus. We further demonstrated that treating the donor mice with palifermin protects against GVHD-associated morbidity, even if the donors are deficient in Valpha14i natural killer T cells. Our findings clearly show that palifermin mediates immunoregulatory effects in addition to its cytoprotective effects and that both are likely to be involved in the mechanism through which palifermin provides protection from acute murine GVHD.

Effect of palifermin in a murine model of graft-versus-host disease (GVHD) associated with Th2 cytokine production, autoantibody production, and glomerulonephritis.

Palifermin (recombinant human keratinocyte growth factor) prevents the development of acute, lethal graft-versus-host disease (GVHD). It does so, at least in part, by protecting cells from injury. Another property of Palifermin is immune regulation. How the latter influences the evolution of GVHD remains uncertain. We explored the effect of Palifermin on GVHD in the DBA/2 --> ((DBA/2)x(C57BL/6))F(1)-hybrid strain combination, a model associated with autoantibody production and glomerulonephritis. Untreated recipients survived until at least day 150 post-induction. Palifermin-treated recipients succumbed between days 50 and 90 with levels of proteinuria of up to 20 g/L, ascites, and rapidly progressive, crescentic glomerulonephritis that was most severe in mice with the greatest levels of proteinuria. Kidney sections from both Palifermin-treated and untreated recipients showed the presence of granular deposits of IgG, IgM, IgA, and C3 in the mesangium and the glomerular basement membrane. Electron microscopy confirmed the extensive glomerular immune complex deposition. Antinuclear and anti-dsDNA antibodies were present in sera from both treated and untreated recipients; however, those in the latter were only detectable if the serum was kept at 37 degrees C, indicating that they were cryoglobulins. IL-4 was detectable only in cultures from Palifermin-treated recipients and the levels of IL-5 and IL-13 were significantly higher in the Palifermin-treated group than in untreated GVHD mice. IFN-gamma was only detectable in untreated GVHD mice. These data suggest that although Palifermin can protect mice with acute GVHD, it exacerbates GVHD in a model associated with autoantibody production and a preponderance of Th2 cytokines.

Graft-versus-host disease in recipients of grafts from natural killer T cell-deficient (Jalpha281(-/-)) donors.

Valpha14i natural killer T cells (NKT cells) are a CD1-restricted subset of NKT cells that express an invariable Valpha14+ Jalpha281+ alphabeta T-cell receptor. To determine whether the absence of Valpha14i NKT cells from the graft affects the development of acute GVHD, we induced GVH reactions using Jalpha281(-/-) mice as donors in the C57BL/6-->(C57BL/6 x DBA/2)F1-hybrid strain combination. Recipients of grafts from Jalpha281(-/-) donors were not protected from either the morbidity or the severe wasting syndrome associated with the development of acute GVHD, but the concentrations of some T helper 1 (Th1) and Th2 cytokines were different from those seen in recipients of grafts from wild-type donors. Interferon-gamma was seen earlier (day 4) in recipients of grafts from Jalpha281(-/-) donors but did not reach the concentrations seen in recipients of grafts from wild-type donors on day 8 (P < 0.02). On day 8, the amount of tumour necrosis factor-alpha released into the serum following the injection of a small amount of lipopolysaccharide was lower in recipients of grafts from Jalpha281(-/-) donors (P < 0.02). The amount of interleukin (IL)-5 was also lower in recipients of grafts from Jalpha281(-/-) donors, when compared to the concentration seen in recipients of grafts from wild-type donors (P < 0.002). IL-13 was seen in recipients of grafts from Jalpha281(-/-) donors but not in recipients of grafts from wild-type donors. Our findings show that the absence of donor Valpha14i NKT cells is associated with lower concentrations of some Th1 cytokines. We also observed higher IL-13 concentrations and lower IL-5 concentrations in recipients of grafts from Jalpha281(-/-) donors indicating a variable effect on Th2 cytokine production.

Surveillance biopsies are superior to functional studies for the diagnosis of acute and chronic renal allograft pathology in children.

In this report of our 3-yr protocol biopsy program, we describe the evolution of acute rejection (AR) and chronic renal allograft nephropathy (CAN) in a cohort of 21 children treated with antibody induction, tacrolimus, mycophenolate mofetil, and prednisone. The aims of this study were to compare the pathogenicity of clinical acute rejection (CAR) and subclinical acute rejection (SAR), and to determine whether functional studies accurately represent acute and chronic renal allograft pathology in pediatric recipients with disproportionately large grafts. Using concurrent biopsies, we evaluated: (i) the utility of changes in the baseline sCr (DeltasCr) to predict both the onset of AR and the response to immunosuppressive therapy; and (ii) the relationship of the calculated creatinine clearance and the presence of pathologic proteinuria to the severity of CAN. We performed 112 biopsies: 11 donor, 73 protocol, 16 acute graft dysfunction and 12 1-month follow-up AR therapy. CAR and SAR were similar in incidence, timing and histologic severity. Progression of CAN was associated with the first episode of CAR (p < 0.02) and the cumulative number of episodes of CAR (p < 0.01), SAR (p < 0.05), CAR plus SAR (p < 0.002) and borderline SAR (B-SAR) (p < 0.006). One-month post-treatment DeltasCrs could not distinguish 1-month follow-up biopsies with histologically confirmed worsened or unchanged AR from those with improved histology (35.2 +/- 74.8% vs. 23.8 +/- 24.9%, p = NS). These findings led to the addition of anti-lymphocyte antibody therapy in five of 10 (50%) cases. Despite 100% 3-yr actuarial graft survival and excellent function (GFR = 111 +/- 36 mL/min/1.73 m(2)), 18 of 21 (86%) patients had grade I CAN or greater chronic histology at a mean +/- sd follow-up period of 18.2 +/- 13.1 months. Thirteen of 21 (62%) patients progressed to grade I CAN at 5.2 +/- 3.6 months and five (38%) of these patients progressed to grade II CAN at 17.8 +/- 11.3 months. Schwartz GFR did not differ between patients with or without CAN (108 +/- 38 mL/min/1.73 m(2) vs. 127 +/- 8 mL/min/1.73 m(2), p = NS). In biopsies with CAN and no associated AR, neither the Banff chronic tubulointerstitial (Banff ci) score nor the Banff chronic grade correlated with the GFR. Proteinuria was not associated with CAN. Clinical AR and SAR are similar histologic lesions with a capacity for CAN progression. In pediatric renal transplant recipients, longitudinal protocol biopsies are superior to functional studies for the diagnosis and post-therapeutic monitoring of AR and for the surveillance of CAN.

Effect of recombinant human keratinocyte growth factor (rHuKGF) on the immunopathogenesis of intestinal graft-vs.-host disease induced without a preconditioning regimen.

We studied the effect of rHuKGF on acute, lethal graft- vs.-host disease (GVHD) in the C57BL/6-->(C57BL/6 X DBA/2)F(1)-hybrid model. rHuKGF-treated recipients did not develop intestinal GVHD despite elevated levels of intestinal NO and TNF alpha, did not develop endotoxemia, and did not die. LPS augmented serum TNF alpha release and intestinal NO production, but did not induce intestinal epithelial cell apoptosis, a phenomenon associated with acute GVHD. These data suggest that KGF prevents the development of acute lethal GVHD by protecting epithelial cell injury mediated by TNF-alpha, NO, and other potential cytotoxic factors. We noted a moderate reduction in intestinal KGFR mRNA expression in untreated GVH mice on day 8, when IFN-gamma mRNA levels were highest. This reduction in KGFR mRNA levels was not seen in recipients of IFN-gamma gene knockout grafts, suggesting that IFN-gamma may be involved in reducing KGFR mRNA expression in the intestine. A similar reduction in intestinal KGFR mRNA expression was also seen in rHuKGF-treated recipients, suggesting that rHuKGF does not mediate its protective effect by maintaining KGFR at control levels. KGF-treatment also redirected the cytokine response in acute GVH mice from Th1 to a mixed pattern of both Th1 and Th2 cytokines. This was associated with histopathologic changes resembling chronic GVHD.

The role of interferon-gamma, nitric oxide and lipopolysaccharide in intestinal graft-versus-host disease developing in F1-hybrid mice.

(C57BL/6 x DBA/2)F1-hybrid mice injected with lymphoid cells from wild-type, C57BL/6 donors develop acute, lethal graft-versus-host disease (GVHD) in which the intestine is a major target. In its destructive phase intestinal GVHD is characterized by apoptosis of intestinal crypt epithelial cells and the development of endotoxaemia. Injection of as little as 10 microg endotoxin is lethal in mice with acute GVHD, and associated with the release of large amounts of tumour necrosis factor-alpha (TNF-alpha) into the serum. To explore the role of interferon-gamma (IFN-gamma) in the pathogenesis of intestinal GVHD we used IFN-gamma gene knockout (gko) mice as donors. Recipients of grafts from these donors did not develop intestinal GVHD and, unlike recipients of wild-type grafts, did not die when injected with lipopolysaccharide (LPS). We also found that injection 10 microg LPS into recipients of wild-type grafts induced apoptosis of intestinal epithelial crypt cells and was associated with a burst of nitric oxide production in the intestine. Administration of N(omega)nitro L-arginine methyl ester blocked this response. In contrast, LPS did not induce either intestinal epithelial cell apoptosis or increased nitric oxide production in recipients of IFN-gamma gko grafts. These findings indicate that donor-derived IFN-gamma is instrumental for the development of intestinal GVHD. In a previous study we showed that recipients of IFN-gamma gko grafts develop high levels of LPS-induced TNF-alpha release. When our current data are viewed in the context of this observation, they suggest that intestinal epithelial cell apoptosis in the parent-->F1-hybrid model of acute GVHD is mediated primarily by nitric oxide rather than TNF-alpha, and that this depends on donor-derived IFN-gamma.

Murine graft-versus-host disease induced using interferon-gamma-deficient grafts features antibodies to double-stranded DNA, T helper 2-type cytokines and hypereosinophilia.

Acute, lethal graft-versus-host disease (GvHD) develops in B6D2F1 hybrid recipients of wild-type, C57BL/6, parental strain grafts; however, when interferon-gamma (IFN-gamma) gene knockout (gko) donors are used, the disease is prolonged and associated with a higher level of engraftment, particularly of T cells. Lesions containing large, mixed cellular infiltrates develop in the skin, liver, pancreas, salivary gland, lung and kidney. In our current study, we wished to determine whether GvHD features a preponderance of T helper 2 (Th2) cytokines in the absence of donor-derived IFN-gamma, and whether autoantibody production, commonly associated with chronic GvHD, also occurs. Because mitogen responsiveness is consistently suppressed in mice with acute GvHD, we wished to measure this response in recipients of IFN-gamma gko grafts. Our findings indicate that spleen cells from the latter produce interleukin (IL)-4, IL-5 and IL-13 in culture, but respond poorly to concanavalin A (Con A) and lipopolysaccharide (LPS). Their sera contain anti-nuclear antibodies (ANA), some of which are specific for double-stranded (ds)DNA and are predominantly immunoglobulin (Ig)M and IgG1. We also noted the presence of numerous eosinophils in the infiltrates developing within the target organs. In some respects, this syndrome bears resemblance to both systemic lupus erythematosus (SLE) and chronic GvHD. However, histological evidence of glomerulonephritis is lacking and proteinuria fails to develop in recipients of IFN-gamma gko grafts, suggesting that IFN-gamma may be necessary for the development of lupus nephritis. On a broader scope, our findings underscore the importance of IFN-gamma in the pathogenetic mechanism of GvHD, and demonstrate that the absence of this cytokine promotes the development of chronic GvHD and autoimmunity.