Revisiting the transcriptional analysis of primary tumours and associated nodal metastases with enhanced biological and statistical controls: application to thyroid cancer.

BACKGROUND: Transcriptome profiling has helped characterise nodal spread. The interpretation of these data, however, is not without ambiguities. METHODS: We profiled the transcriptomes of papillary thyroid cancer nodal metastases, associated primary tumours and primary tumours from N0 patients. We also included patient-matched non-cancerous thyroid and lymph node samples as controls to address some limits of previous studies. RESULTS: The transcriptomes of patient-matched primary tumours and metastases were more similar than those of unrelated metastases/primary pairs, as previously reported in other organ systems. This similarity partly reflected patient background. Lymphoid tissues in the metastases confounded the comparison of patient-matched primary tumours and metastases. We circumvented this with an original data adjustment, revealing a differential expression of stroma-related gene signatures also regulated in other organs. The comparison of N0 vs N+ primary tumours uncovered a signal irreproducible across independent data sets. This signal was also detectable when comparing the non-cancerous thyroid tissues adjacent to N0 and N+ tumours, suggesting a cohort-specific bias also likely present in previous similarly sized studies. Classification of N0 vs N+ yielded an accuracy of 63%, but additional statistical controls absent in previous studies revealed that this is explainable by chance alone. We used large data sets from The Cancer Genome Atlas: N0 vs N+ classification was not better than random for most cancers. Yet, it was significant, but of limited accuracy (<70%) for thyroid, breast and head and neck cancers. CONCLUSIONS: The clinical potential of gene expression to predict nodal metastases seems limited for most cancers.

Systems biology of cancer: entropy, disorder, and selection-driven evolution to independence, invasion and "swarm intelligence".

Our knowledge of the biology of solid cancer has greatly progressed during the last few years, and many excellent reviews dealing with the various aspects of this biology have appeared. In the present review, we attempt to bring together these subjects in a general systems biology narrative. It starts from the roles of what we term entropy of signaling and noise in the initial oncogenic events, to the first major transition of tumorigenesis: the independence of the tumor cell and the switch in its physiology, i.e., from subservience to the organism to its own independent Darwinian evolution. The development after independence involves a constant dynamic reprogramming of the cells and the emergence of a sort of collective intelligence leading to invasion and metastasis and seldom to the ultimate acquisition of immortality through inter-individual infection. At each step, the probability of success is minimal to infinitesimal, but the number of cells possibly involved and the time scale account for the relatively high occurrence of tumorigenesis and metastasis in multicellular organisms.

A gene expression signature distinguishes normal tissues of sporadic and radiation-induced papillary thyroid carcinomas.

BACKGROUND: Papillary thyroid cancer (PTC) incidence increased dramatically in children after the Chernobyl accident, providing a unique opportunity to investigate the molecular features of radiation-induced thyroid cancer. In contrast to the previous studies that included age-related confounding factors, we investigated mRNA expression in PTC and in the normal contralateral tissues of patients exposed and non-exposed to the Chernobyl fallout, using age- and ethnicity-matched non-irradiated cohorts. METHODS: Forty-five patients were analysed by full-genome mRNA microarrays. Twenty-two patients have been exposed to the Chernobyl fallout; 23 others were age-matched and resident in the same regions of Ukraine, but were born after 1 March 1987, that is, were not exposed to ¹³¹I. RESULTS: A gene expression signature of 793 probes corresponding to 403 genes that permitted differentiation between normal tissues from patients exposed and from those who were not exposed to radiation was identified. The differences were confirmed by quantitative RT-PCR. Many deregulated pathways in the exposed normal tissues are related to cell proliferation. CONCLUSION: Our results suggest that a higher proliferation rate in normal thyroid could be related to radiation-induced cancer either as a predisposition or as a consequence of radiation. The signature allows the identification of radiation-induced thyroid cancers.

A general method to derive robust organ-specific gene expression-based differentiation indices: application to thyroid cancer diagnostic.

Differentiation is central to development, while dedifferentiation is central to cancer progression. Hence, a quantitative assessment of differentiation would be most useful. We propose an unbiased method to derive organ-specific differentiation indices from gene expression data and demonstrate its usefulness in thyroid cancer diagnosis. We derived a list of thyroid-specific genes by selecting automatically those genes that are expressed at higher level in the thyroid than in any other organ in a normal tissue's genome-wide gene expression compendium. The thyroid index of a tissue was defined as the median expression of these thyroid-specific genes in that tissue. As expected, the thyroid index was inversely correlated with meta-PCNA, a proliferation metagene, across a wide range of thyroid tumors. By contrast, the two indices were positively correlated in a time course of thyroid-stimulating hormone (TSH) activation of primary thyrocytes. Thus, the thyroid index captures biological information not integrated by proliferation rates. The differential diagnostic of follicular thyroid adenomas and follicular thyroid carcinoma is a notorious challenge for pathologists. The thyroid index discriminated them as accurately as did machine-learning classifiers trained on the genome-wide cancer data. Hence, although it was established exclusively from normal tissue data, the thyroid index integrates the relevant diagnostic information contained in tumoral transcriptomes. Similar results were obtained for the classification of the follicular vs classical variants of papillary thyroid cancers, that is, tumors dedifferentiating along a different route. The automated procedures demonstrated in the thyroid are applicable to other organs.

Gene expression profiles for radiation-induced thyroid cancer.

The question whether radiation-induced thyroid cancer differs by its molecular biology from sporadic disease still remains. Studies on tissue from patients who developed thyroid cancer after the Chernobyl accident have provided a unique opportunity to look for biological consequences of low-dose irradiation by comparing the gene expression profile of sporadic papillary thyroid cancer (PTC), whose aetiology is unknown, and PTC induced by internal radiation. So far, four transcriptomic studies comparing radiation-induced and sporadic thyroid cancer have been reported. However, no final conclusion has been drawn regarding the presence of a radiation signature, as either no difference was noted or the reported differences were not sufficiently convincing due to the low number of cases analysed or to the presence of confounding factors. The list of putative biological and clinical factors that may influence the PTC gene expression profile is long, but there are sufficient data reported in the literature to link expression profiles with differing pathological variants of PTC. The comparison of expression profiles in the tumour samples allows the search for a radiation signature, whereas the comparison of expression profiles of the normal contralateral tissues offers a substantial opportunity for assessing the existence of a susceptibility to radiation that could be responsible for tumour development. We have undertaken this analysis as part of a European Union-funded project, GENRISK-T. Gene expression profiles were investigated in tumours that have arisen in the population exposed to fallout from Chernobyl (i.e. born before 26 April 1986) and were compared with profiles of tumours of similar pathology arising in an age-matched population, residing in the same geographical area (same ethnicity) and born after 1 January 1987. RNA samples from these tumours and their contralateral normal tissues were obtained from the Chernobyl Tissue Bank. Several lines of evidence suggest that the predisposition to developing cancer after radiation exposure is variable in the general population and may be measurable from gene expression.

Human cancer cell lines: Experimental models for cancer cells in situ? For cancer stem cells?

Established human cancer cell lines are routinely used as experimental models for human cancers. Their validity for such use is analyzed and discussed, with particular focus on thyroid tumors. Although cell lines retain some properties of the cells of origin, from the points of view of their genetics, epigenetics and gene expression, they show clear differences in these properties compared to in vivo tumors. This can be explained by a prior selection of initiating cells and a Darwinian evolution in vitro. The properties of the cell lines are compared to those of the postulated cancer stem cells and their use as models in this regard are discussed. Furthermore, other proper and possible uses of the cell lines are discussed.

[Transcription markers of survival in breast neoplasms measuring the signals from the proliferation]. / Les marqueurs transcriptionnels de la survie dans le cancer du sein mesurent des signaux liés à la prolifération.

We show that proliferation-related signals are omnipresent in the breast cancer transcriptome. As a result, many transcriptional signatures generated at random are valuable for the prognosis of disease-free survival: despite their biological rationale, 30-60% of published prognostic signatures are not significantly better. We propose a mathematical transformation, the super PCNA decovolution, which removes proliferation-related signals from tumours transcriptional profiles. Both random and published signatures loose nearly all their prognostic value after removal of these signals.

Genome-wide gene expression profiling suggests distinct radiation susceptibilities in sporadic and post-Chernobyl papillary thyroid cancers.

Papillary thyroid cancers (PTCs) incidence dramatically increased in the vicinity of Chernobyl. The cancer-initiating role of radiation elsewhere is debated. Therefore, we searched for a signature distinguishing radio-induced from sporadic cancers. Using microarrays, we compared the expression profiles of PTCs from the Chernobyl Tissue Bank (CTB, n=12) and from French patients with no history of exposure to ionising radiations (n=14). We also compared the transcriptional responses of human lymphocytes to the presumed aetiological agents initiating these tumours, gamma-radiation and H(2)O(2). On a global scale, the transcriptomes of CTB and French tumours are indistinguishable, and the transcriptional responses to gamma-radiation and H(2)O(2) are similar. On a finer scale, a 118 genes signature discriminated the gamma-radiation and H(2)O(2) responses. This signature could be used to classify the tumours as CTB or French with an error of 15-27%. Similar results were obtained with an independent signature of 13 genes involved in homologous recombination. Although sporadic and radio-induced PTCs represent the same disease, they are distinguishable with molecular signatures reflecting specific responses to gamma-radiation and H(2)O(2). These signatures in PTCs could reflect the susceptibility profiles of the patients, suggesting the feasibility of a radiation susceptibility test.

Roles of hydrogen peroxide in thyroid physiology and disease.

CONTEXT: The long-lived thyroid cell generates, for the synthesis of thyroid hormones, important amounts of H2O2 that are toxic in other cell types. This review analyzes the protection mechanisms of the cell and the pathological consequences of disorders of this system. EVIDENCE ACQUISITION: The literature on H2O2 generation and disposal, thyroid hormone synthesis, and their control in the human thyroid is analyzed. EVIDENCE SYNTHESIS: In humans, H2O2 production by dual-oxidases and consequently thyroid hormone synthesis by thyroperoxidase are controlled by the phospholipase C-Ca2+-diacylglycerol arm of TSH receptor action. H2O2 in various cell types, and presumably in thyroid cells, is a signal, a mitogen, a mutagen, a carcinogen, and a killer. The various protection mechanisms of the thyroid cell against H2O2 are analyzed. They include the separation of the generating enzymes (dual-oxidases), their coupling to thyroperoxidase in a proposed complex, the thyroxisome, and H2O2 degradation systems. CONCLUSIONS: It is proposed that various pathologies can be explained, at least in part, by overproduction and lack of degradation of H2O2 (tumorigenesis, myxedematous cretinism, and thyroiditis) and by failure of the H2O2 generation or its positive control system (congenital hypothyroidism).

Gene expression and the biological phenotype of papillary thyroid carcinomas.

The purpose of this paper is to correlate the molecular phenotype of papillary thyroid carcinoma (PTC) to their biological pathology. We hybridized 26 PTC on microarrays and showed that nearly 44% of the transcriptome was regulated in these tumors. We then combined our data set with two published PTC microarray studies to produce a platform- and study-independent list of PTC-associated genes. We further confirmed the mRNA regulation of 15 genes from this list by quantitative reverse transcription-PCR. Analysis of this list with statistical tools led to several conclusions: (1) there is a change in cell population with an increased expression of genes involved in the immune response, reflecting lymphocyte infiltration in the tumor compared to the normal tissue. (2) The c-jun N-terminal kinase pathway is activated by overexpression of its components. (3) The activation of ERKK1/2 by genetic alterations is supplemented by activation of the epidermal growth factor but not of the insulin-like growth factor signaling pathway. (4) There is a downregulation of immediate early genes. (5) We observed an overexpression of many proteases in accordance with tumor remodeling, and suggested a probable role of S100 proteins and annexin A2 in this process. (6) Numerous overexpressed genes favor the hypothesis of a collective migration mode of tumor cells.

Absence of a specific radiation signature in post-Chernobyl thyroid cancers.

Thyroid cancers have been the main medical consequence of the Chernobyl accident. On the basis of their pathological features and of the fact that a large proportion of them demonstrate RET-PTC translocations, these cancers are considered as similar to classical sporadic papillary carcinomas, although molecular alterations differ between both tumours. We analysed gene expression in post-Chernobyl cancers, sporadic papillary carcinomas and compared to autonomous adenomas used as controls. Unsupervised clustering of these data did not distinguish between the cancers, but separates both cancers from adenomas. No gene signature separating sporadic from post-Chernobyl PTC (chPTC) could be found using supervised and unsupervised classification methods although such a signature is demonstrated for cancers and adenomas. Furthermore, we demonstrate that pooled RNA from sporadic and chPTC are as strongly correlated as two independent sporadic PTC pools, one from Europe, one from the US involving patients not exposed to Chernobyl radiations. This result relies on cDNA and Affymetrix microarrays. Thus, platform-specific artifacts are controlled for. Our findings suggest the absence of a radiation fingerprint in the chPTC and support the concept that post-Chernobyl cancer data, for which the cancer-causing event and its date are known, are a unique source of information to study naturally occurring papillary carcinomas.

Evolutionary and immunological implications of contemporary HIV-1 variation.

Evolutionary modelling studies indicate less than a century has passed since the most recent common ancestor of the HIV-1 pandemic strains and, in that time frame, an extraordinarily diverse viral population has developed. HIV-1 employs a multitude of schemes to generate variants: accumulation of base substitutions, insertions and deletions, addition and loss of glycosylation sites in the envelope protein, and recombination. A comparison between HIV and influenza virus illustrates the extraordinary scale of HIV variation, and underscores the importance of exploring innovative HIV vaccine strategies. Deeper understanding of the implications of variation for both antibody and T-cell responses may help in the effort to rationally design vaccines that stimulate broad cross-reactivity. The impact of HIV-1 variation on host immune response is reviewed in this context.

The paradox of alloreactivity and self MHC restriction: quantitative analysis and statistics.

Although 1-24% of T cells are alloreactive, i.e., respond to MHC molecules encoded by a foreign haplotype, it is generally believed that T cells cannot recognize foreign peptides binding foreign MHC molecules. We show using a quantitative model that, if T cell selection and activation are affinity-driven, then an alloreactivity of 1-24% is incompatible with the textbook notion that self MHC restriction is absolute. If an average of 1% of clones are alloreactive, then according to our model, at most 20-fold more clones should, on average, be activated by antigens presented on self MHC than by antigens presented on foreign MHC. This ratio is at best 5 if alloreactivity is 5%. These results describe average properties of the murine immune system, but not the outcome of individual experiments. Using supercomputer technology, we simulated 100,000 MHC restriction experiments. Although the average restriction ratio was 7.1, restriction was absolute in 10% of the simulated experiments, greater than 100, although not absolute, in 29%, and below 6 in 24%. This extreme variability agrees with experimental estimates. Our analysis suggests that alloreactivity and average self MHC restriction both cannot be high, but that a low average restriction level is compatible with high levels in a significant number of experiments.

Deriving quantitative constraints on T cell selection from data on the mature T cell repertoire.

The T cell repertoire is shaped in the thymus through positive and negative selection. Thus, data about the mature repertoire may be used to infer information on how TCR generation and selection operate. Assuming that T cell selection is affinity driven, we derive the quantitative constraints that the parameters driving these processes must fulfill to account for the experimentally observed levels of alloreactivity, self MHC restriction and the frequency of cells recognizing a given foreign Ag. We find that affinity-driven selection is compatible with experimental estimates of these latter quantities only if 1) TCRs see more peptide residues than MHC polymorphic residues, 2) the majority of positively selected clones are deleted by negative selection, 3) between 1 and 3.6 clonal divisions occur on average in the thymus after completion of TCR rearrangement, and 4) selection is driven by 103-105 self peptides.

A quantitative theory of affinity-driven T cell repertoire selection.

Binding of the T cell antigen receptor (TCR) to peptides presented on molecules encoded by major histocompatibility complex (MHC) genes is the key event driving T cell development and activation. Selection of the T cell repertoire in the thymus involves two steps. First, positive selection promotes the survival of cells binding thymic self-MHC-peptide complexes with sufficient affinity. The resulting repertoire is self-MHC restricted: it recognizes foreign peptides presented on self, but not foreign MHC. Second, negative selection deletes cells which may be potentially harmful because their receptors interact with self-MHC-peptide complexes with too high an affinity. The mature repertoire is also highly alloreactive: a large fraction of T cells respond to tissues harboring foreign MHC. We derive mathematical expressions giving the frequency of alloreactivity, the level of self-MHC restriction, and the fraction of the repertoire activated by a foreign peptide, as a function of the parameters driving the generation and selection of the repertoire: self-MHC and self-peptide diversity, the stringencies of positive and negative selection, and the number of peptide and MHC polymorphic residues that contribute to T cell receptor binding. Although the model is based on a simplified digit string representation of receptors, all the parameters but one relate directly to experimentally determined quantities. The only parameter without a biological counterpart has no effect on the model's behavior besides a trivial and easily preventable discretization effect. We further analyse the role of the MHC and peptide contribution to TCR binding, and find that their relative, rather than absolute value, is important in shaping the mature repertoire. This result makes it possible to adopt different physical interpretations for the digit string formalism. We also find that the alloreactivity level can be inferred directly from data on the stringency of selection, and that, in agreement with recent experiments, it is not affected by thymic selection.

Explaining high alloreactivity as a quantitative consequence of affinity-driven thymocyte selection.

Interactions between alphabeta T cell receptors and peptides bound to molecules encoded by the MHC genes underly T cell activation. More than 1% of T cells are activated by foreign (allogenic) MHC molecules, a phenomenon called alloreactivity. Reconciling the high frequency of alloreactivity with the fact that only 1 T cell in 10(4)-10(6) responds to a given foreign antigen presented on self MHC has been a long-standing puzzle. We show, by using a quantitative model, that this difference follows from the affinity model of T cell selection. Further, we demonstrate that highly alloreactive pre- and post-selection repertoires can be obtained without assuming germline bias of T cell receptors toward recognition of allele-specific MHC residues. It has been proposed that alloreactivity occurs because self and foreign MHCs bind different subsets of self peptides or alter their conformation differently. We find that such effects decrease rather than increase alloreactivity. Overall, our results show that the affinity model of T cell selection can quantitatively explain both self MHC restriction and high alloreactivity.

Size and connectivity of the idiotypic network are independent of the discreteness of the affinity distribution.

Idiotypic interactions may be a factor in the selection of the B cell repertoire. Simulations of an evolving idiotypic network where new clones are introduced on a daily basis have shown that macroscopic properties, such as network size and connectivity, attain stationary values despite the rapid turnover of individual clones, indicating that idiotypic networks possess self-organizing properties. Affinities between antibodies were either zero, low (0.1), or high (1.0) in these simulations. It has been suggested that network properties may change when affinities take arbitrary real values. Here we show that the previous results of De Boer & Perelson on network size and connectivity are not changed when affinities take many different, closely spaced values.

Development of an idiotypic network in shape space.

Based upon the shape-space formalism, a model of an idiotypic network including both bound and free immunoglobulins is simulated. Our point of interest is the network development in the context of self antigens. The investigations are organized around simulations initiated by various spatial configurations of antigens; the behavior of the system with respect to antigens is analyzed in terms of morphogenetic processes occurring in the shape space. For certain values of the parameters, the network expands by traveling waves. The resulting spatial pattern is a partition of the shape space into zones where introduction of an antigen entails an infinite growth of the clones binding to it, and into zones where, on the contrary, the anti-antigen idiotypes decrease. Among the parameter combinations tested, some produce a partition that remains static whereas others produce a partition that changes in time. For other values of the parameters, the patterns generated do not partition shape space into zones; in these cases, it is observed that the system systematically explodes when an antigen is present.