A 50-gene signature is a novel scoring system for tumor-infiltrating immune cells with strong correlation with clinical outcome of stage I/II non-small cell lung cancer

Purpose: To identify a novel system for scoring intratumoral immune response that can improve prognosis and therapy decisions in early stage non-small cell lung cancer (NSCLC). Methods/patients: Eighty-four completely resected stage I/II NSCLC without adjuvant therapy were classified by expression profiling using whole genome microarrays. An external cohort of 162 tumors was used to validate the results. Immune cells present in tumor microenvironment were evaluated semiquantitatively by CD20, CD79, CD3, CD8, CD4 and CD57 immunostaining. Univariate and multivariate analyses of variables associated with recurrence-free survival were performed. Results: Initial molecular classification identified three clusters, one with significantly better RFS. A reduced two-subgroup classification and a 50-gene predictor were built and validated in an external dataset: high and low risk of recurrence patients (HR = 3.44; p = 0.001). Analysis of the predictor´s genes showed that the vast majority were related to a B/plasma cell immune response overexpressed in the low-risk subgroup. The predictor includes genes coding for unique B lineage-specific genes, functional elements or other genes that, although non-restricted to this lineage, have strong influence on B-cell homeostasis. Immunostains confirmed increased B-cells in the low-risk subgroup. Gene signature (p < 0.0001) and CD20 (p < 0.05) were predictors for RFS, while CD79 and K-RAS mutations showed a tendency. Conclusions: Favorable prognosis in completely resected NSCLC is determined by a B-cell-mediated immune response. It can be differently scored by a 50-gene expression profile or by CD20 immunostaining. That prognosis information not reflected by traditional classifications may become a new tool for determining individualized adjuvant therapies (AU)

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A 50-gene signature is a novel scoring system for tumor-infiltrating immune cells with strong correlation with clinical outcome of stage I/II non-small cell lung cancer.

PURPOSE: To identify a novel system for scoring intratumoral immune response that can improve prognosis and therapy decisions in early stage non-small cell lung cancer (NSCLC). METHODS/PATIENTS: Eighty-four completely resected stage I/II NSCLC without adjuvant therapy were classified by expression profiling using whole genome microarrays. An external cohort of 162 tumors was used to validate the results. Immune cells present in tumor microenvironment were evaluated semiquantitatively by CD20, CD79, CD3, CD8, CD4 and CD57 immunostaining. Univariate and multivariate analyses of variables associated with recurrence-free survival were performed. RESULTS: Initial molecular classification identified three clusters, one with significantly better RFS. A reduced two-subgroup classification and a 50-gene predictor were built and validated in an external dataset: high and low risk of recurrence patients (HR = 3.44; p = 0.001). Analysis of the predictor´s genes showed that the vast majority were related to a B/plasma cell immune response overexpressed in the low-risk subgroup. The predictor includes genes coding for unique B lineage-specific genes, functional elements or other genes that, although non-restricted to this lineage, have strong influence on B-cell homeostasis. Immunostains confirmed increased B-cells in the low-risk subgroup. Gene signature (p < 0.0001) and CD20 (p < 0.05) were predictors for RFS, while CD79 and K-RAS mutations showed a tendency. CONCLUSIONS: Favorable prognosis in completely resected NSCLC is determined by a B-cell-mediated immune response. It can be differently scored by a 50-gene expression profile or by CD20 immunostaining. That prognosis information not reflected by traditional classifications may become a new tool for determining individualized adjuvant therapies.

Pancreatic homeodomain transcription factor IDX1/IPF1 expressed in developing brain regulates somatostatin gene transcription in embryonic neural cells.

Hox-like homeodomain proteins play a critical role during embryonic development by regulating the transcription of genes that are important for the generation of specific organs or cell types. The homeodomain transcription factor IDX1/IPF1, the expression of which was thought until recently to be restricted to the pancreas and foregut, is required for pancreas development and for the expression of genes controlling glucose homeostasis. We report that IDX1/IPF1 is also expressed in embryonic rat brain at a time coincident with active neurogenesis. Electrophoretic mobility shift assays with nuclear extracts of embryonic brains indicated that IDX1/IPF1 binds to two somatostatin promoter elements, SMS-UE-B and the recently discovered SMS-TAAT3. The requirement of these elements for IDX1/IPF1 transactivation of the somatostatin gene in neural cells was confirmed in transfection studies using embryonic cerebral cortex-derived RC2.E10 cells. Immunohistochemical staining of rat embryos showed IDX1/IPF1-positive cells located near the ventricular surface in germinative areas of the developing central nervous system. Cellular colocalization of IDX1/IPF1 and somatostatin was found in several areas of the developing brain, including cortex, ganglionic eminence, hypothalamus, and inferior colliculus. These results support the notion that IDX1/IPF1 regulates gene expression during development of the central nervous system independent of its role on pancreas development and function.

The pancreatic homeodomain transcription factor IDX1/IPF1 is expressed in neural cells during brain development.

Expression of the homeodomain transcription factor IDX1/IPF1 has been shown to be restricted to cells in the developing foregut that form the pancreatic primordium. In the adult, IDX1/IPF1 is expressed in the duodenum and pancreatic islets. The IDX1/IPF1 gene is required for pancreatic development, and in the human, heterozygous mutations have been linked to diabetes mellitus. In the present communication, we report that IDX1/IPF1 is expressed in discrete cells of the rat central nervous system during embryonic development. Using RT-PCR, IDX1/IPF1 mRNA was detected in neural precursor RC2.E10 cells, as well as in both forebrain and hindbrain of developing rats at embryonic day 15 (E15). The presence of IDX1/IPF1 protein was confirmed by Western immunoblotting. Immunohistochemical analyses of sagittal sections of E15 rats demonstrated the presence of scattered IDX1/IPF1-immunopositive cells in the forebrain. Finally, electrophoretic mobility shift assays using nuclear extracts from neural cells revealed the presence of IDX1/IPF1 bound to a putative homeodomain protein DNA-binding site present in the promoter of the glial fibrillary acidic protein gene. Our results suggest that IDX1/IPF1 may have previously unsuspected extrapancreatic functions during development of neural cells in the central nervous system.

Synthesis and differentially regulated processing of proinsulin in developing chick pancreas, liver and neuroretina.

Regulated preproinsulin gene expression in nonpancreatic tissues during development has been demonstrated in rodents, Xenopus and chicken. Little is known, however, about the synthesis and processing of the primary protein product, proinsulin, in comparison with these events in pancreas. Using specific antisera and immunocytochemistry, immunoblot and HPLC criteria, we characterize the differential processing of proinsulin in developing neuroretina, liver and pancreas. The chick embryo pancreas expresses the convertase PC2, and largely processes proinsulin to insulin. In contrast, little or no mature PC2 is present in embryonic liver and neuroretina and the (pro)insulin immunoactivity identified is predominantly proinsulin.

Developmentally regulated expression of the preproinsulin gene in the chicken embryo during gastrulation and neurulation.

Despite the absence of a pancreas, which develops between embryonic day 3 (E3) to E4, previous studies showed that insulin receptors are widely expressed in chicken embryos from the blastoderm stage (unincubated embryo, E0) through gastrulation (E0.5-E1), neurulation (E1.5-E2), and organogenesis. We now characterize prepancreatic preproinsulin gene expression and its regulation, using a highly sensitive modification of the polymerase chain reaction. We found preproinsulin messenger RNA (mRNA) expression at all stages, from the unincubated chicken blastoderm through early organogenesis, with the highest expression in embryos undergoing gastrulation. In situ hybridization analysis of E1-E1.5 embryos in toto showed widespread distribution of preproinsulin mRNA in a pattern similar to that of insulin receptor mRNA. In contrast, insulin-like growth factor-I mRNA expression appeared later than preproinsulin mRNA in the embryo; it was first demonstrable in the head portion of E3 and was found in head, trunk, and caudal regions by E4. With a novel culture system for chicken embryos during neurulation, we examined whether glucose regulated prepancreatic preproinsulin mRNA expression. Embryos cultured in glucose-free medium had increased preproinsulin mRNA with respect to the value in ovo, but the addition of 17 mM glucose had no stimulatory effect. In marked contrast, in organ cultures of E13 pancreas, insulin mRNA expression decreased in glucose-free medium by 50% relative to that in ovo. The addition of glucose restored the levels to a concentration similar to that found in ovo. Exogenous insulin added to cultured E1.5 embryos increased protein and DNA synthesis. We conclude that the preproinsulin gene is widely expressed in chicken embryo structures throughout gastrulation and neurulation. This prepancreatic preproinsulin mRNA is differentially regulated compared to the pancreatic mRNA. Preproinsulin gene products may have a role in cell proliferation, differentiation, or survival in very early avian embryos at a time when insulin-like growth factor-I expression is absent or undetectable.

IGF-I and the IGF-I receptor in development of nonmammalian vertebrates.

Extracellular signals are likely to be involved in the control of growth and differentiation during embryogenesis of vertebrates. These signals include, among others, several members of the insulin family: insulin-like growth factor (IGF)-I, IGF-II, and insulin. In the chick embryo, maternal IGF-I is stored in the yolk. In addition, the embryonic IGF-I gene is expressed very early and in late development in multiple tissues. We have used reverse-transcribed (RT) RNA and amplification by the polymerase chain reaction (PCR) to detect IGF-I gene expression. IGF-I was preferentially expressed in cephalic regions during late neurulation and early organogenesis. During late organogenesis, in some tissues, such as the eye lens, IGF-I gene expression is compartmentalized to a subset of cells, the epithelial cells. In these lens cells, IGF-I stimulates transcription of the delta-crystallin gene. Competence to respond to IGF-I exists in multiple cell types, since, based on binding studies, receptors for IGF-I are widespread in the gastrulating and neurulating embryo. Target tissues in which an autocrine/paracrine role for IGF-I appears more likely are the developing eye lens and retina, which are avascular organs rich in IGF-I receptors. In late development, IGF-I may have an additional endocrine role, with an impact on the general growth of the chick embryo. In embryos developed ex ovo, that show growth retardation after day 10 of embryogenesis, IGF-I serum levels are very low. By day 8, expression of IGF-I mRNA in these embryos is markedly reduced in multiple tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of physiological prolactin levels in growth and prolactin receptor content of prostate glands and testes in developing male rats.

We have investigated the role of physiological prolactin levels in the development of prepubertal male rats. Prolactin GH and testosterone levels, as well as body, ventral prostate and testicular weight, have been analysed in both control and bromocriptine-treated rats between 21 and 60 days of life. Furthermore the role of prolactin in the regulation of its own receptors has also been studied during the same period. In control rats, prolactin levels showed a prepubertal peak of secretion at 25 days of age. At this time GH and testosterone levels were low and did not show any significant variation. After this age, prolactin levels increased more gradually; determinations of GH showed great variation with low levels in most of the rats and very high values in the other animals; testosterone levels remained low until day 35 after which they increased. Simultaneously with the serum prolactin peak on day 25, a decrease in prolactin-binding capacity of ventral prostate glands, was observed and a maximum rate of body, prostate and testicular weight gain was obtained. Furthermore, in rats with pharmacologically suppressed serum prolactin levels (lower than 1 microgram/l), prolactin binding to prostate glands as well as the weight of body, ventral prostate and testes were lower than in control animals. When results were expressed in mg prostate or testes/g body weight, testes from 25-day-old treated rats weighed significantly less than controls. The later stages of development, from days 25 to 60, were characterized by an initial decline in serum prolactin levels at 29 days of age which was followed by a continuous increase until adult values were reached. During this period, prostatic prolactin receptors which were at their lowest value at 33 days of age showed a gradual rise parallel with the observed increase in plasma prolactin levels. When testicular tissue was analysed, no changes in prolactin-binding sites caused by sexual maturation were observed. The present results indicate that physiological prolactin secretion has a specific effect on the normal increase in the prostate, testes and body weight and clearly is also implicated in the regulation of its prostatic receptors at the earlier stages of development.

[Evaluation of free and bound fractions resulting from the interaction of prolactin with its specific receptors]. / Valoración de las fracciones libre y unida resultantes de la interacción de la prolactina con sus receptores específicos.

Binding of either "cold" or 125I-PRL to their specific receptors (fraction after centrifugation at 15,000 and 100,000 X g) obtained from late pregnant rat liver, pre- and post-dissociation with MgCl2, has been studied. Binding was higher with cold hormone (delta 21.63%) than with 125I-PRL. Similarly, binding to the 100,000 X g fraction was also higher than to the 15,000 X g one. Dissociation by MgCl2 improved binding to the 100,000 X g fraction (delta 17.27%), while reduced the 15,000 X g fraction binding (delta 11.71%), underlying the impurity of the latter fraction. Control studies with rLH, rFSH, hACTH, insulin, glucagon and hGH evidenced the specificity of the preparation to bind lactogenic hormones. Binding increases with PRL and receptor concentration, reaching equilibrium between bound PRL/unbound PRL. An amount of PRL unable to bind to the receptor is always present. Even with high receptor concentrations (3,500 micrograms/0.1 ml) there is still about 25% of unbound PRL. When reincubating this previously unbound PRL with a fresh receptor preparation identical to the one used in the first incubation, a similar proportion of bound PRL/unbound PRL is obtained. These results suggest the existence of a heterogeneity in the receptor preparation.