Bridging the gap between animal or in vitro models and human disease is essential in medical research. Researchers often suggest that a biological mechanism is relevant to human cancer from the statistical association of a gene expression marker (a signature) of this mechanism, that was discovered in an experimental system, with disease outcome in humans. We examined this argument for breast cancer. Surprisingly, we found that gene expression signatures—unrelated to cancer—of the effect of postprandial laughter, of mice social defeat and of skin fibroblast localization were all significantly associated with breast cancer outcome. We next compared 47 published breast cancer outcome signatures to signatures made of random genes. Twenty-eight of them (60%) were not significantly better outcome predictors than random signatures of identical size and 11 (23%) were worst predictors than the median random signature. More than 90% of random signatures >100 genes were significant outcome predictors. We next derived a metagene, called meta-PCNA, by selecting the 1% genes most positively correlated with proliferation marker PCNA in a compendium of normal tissues expression. Adjusting breast cancer expression data for meta-PCNA abrogated almost entirely the outcome association of published and random signatures. We also found that, in the absence of adjustment, the hazard ratio of outcome association of a signature strongly correlated with meta-PCNA (R2 = 0.9). This relation also applied to single-gene expression markers. Moreover, >50% of the breast cancer transcriptome was correlated with meta-PCNA. A corollary was that purging cell cycle genes out of a signature failed to rule out the confounding effect of proliferation. Hence, it is questionable to suggest that a mechanism is relevant to human breast cancer from the finding that a gene expression marker for this mechanism predicts human breast cancer outcome, because most markers do. The methods we present help to overcome this problem.

The pituitary hormone thyrotropin stimulates the function, expression of differentiation and growth of thyrocytes by cyclic AMP-dependent mechanisms. Tissue hyperplasia and hyperthyroidism are therefore expected to result when activation of the adenylyl cyclase-cAMP cascade is unregulated. This is observed in several situations, including when somatic mutations impair the GTPase activity of the G protein Gsa (ref 6, 7). Such a mechanism is probably responsible for the development of a minority of monoclonal hyperfunctioning thyroid adenomas. Here we identify somatic mutations in the carboxy-terminal portion of the third cytoplasmic loop of the thyrotropin receptor in three out of eleven hyperfunctioning thyroid adenomas. These mutations are restricted to tumour tissue and involve two different residues (aspartic acid at position 619 to glycine in two cases, and alanine at position 623 to isoleucine in one case). The mutant receptors confer constitutive activation of adenylyl cyclase when tested by transfection in COS cells. This shows that G-protein-coupled receptors are susceptible to constitutive activation by spontaneous somatic mutations and may thus behave as proto-oncogenes.

Two cDNAs encoding NADPH oxidases and constituting the thyroid H 2 O 2 generating system have been cloned. The strategy of cloning was based on the functional similarities between H 2 O 2 generation in leukocytes and the thyroid, according to the hypothesis that one of the components of the thyroid system would belong to the gp91 Phox /Mox1 gene family and display sequence similarities with gp91Phox . Screening at low stringency with a gp91Phox probe of cDNA libraries from thyroid cells in primary culture yielded two distinct human cDNA clones harboring open reading frames of 1551 (ThOX1) and 1548 amino acids (ThOX2), respectively. The encoded polypeptides display 83% sequence similarity and are clearly related to gp91Phox (53 and 47% similarity). The theoretical molecular mass of 177 kDa is close to the apparent molecular mass of 180 kDa of the native corresponding porcine flavoprotein and the protein(s) detected by Western blot in dog and human thyroid. ThOX1 and ThOX2 display sequence similarities of 53% and 61%, respectively, with a predicted protein of Caenorhabditis elegans over their entire length. They show along their first 500 amino acids a similarity of 43% with thyroperoxidase. The corresponding genes of ThOX1 and ThOX2 are closely linked on chromosome 15q15.3. The dog mRNA expression is thyroid-specific and up-regulated by agents activating the cAMP pathway as is the synthesis of the polypeptides they are coding for. In human thyroid the positive regulation by cAMP is less pronounced. The proteins ThOX1 and ThOX2 accumulate at the apical membrane of thyrocytes and are co-localized with thyroperoxidase.

I. CELL PROLIFERATION IN NORMAL THYROID TISSUE cles and of their supporting mesenchymal tissue and IN VIVO cells, the endothelial cells of the capillaries (20%) and the fibroblasts (10%) (78). Scarce calcitonin-secreting A. Physiological Situation parafollicular cells are located at the periphery of the follicles. After its differentiation in the fetus, the tissue The thyroid tissue is mainly composed of thyroid grows roughly in parallel with body weight and remains follicular cells, the thyrocytes (70954, arranged in folli-at the same size throughout adult life.