Intratumor heterogeneity in evolutionary models of tumor progression

With rare exceptions, human tumors arise from single cells that have accumulated the necessary number and types of heritable alterations. Each such cell leads to dysregulated growth and eventually the formation of a tumor. Despite their monoclonal origin, at the time of diagnosis most tumors show a striking amount of intratumor heterogeneity in all measurable phenotypes; such heterogeneity has implications for diagnosis, treatment efficacy, and the identification of drug targets. An understanding of the extent and evolution of intratumor heterogeneity is therefore of direct clinical importance. In this article, we investigate the evolutionary dynamics of heterogeneity arising during exponential expansion of a tumor cell population, in which heritable alterations confer random fitness changes to cells. We obtain analytical estimates for the extent of heterogeneity and quantify the effects of system parameters on this tumor trait. Our work contributes to a mathematical understanding of intratumor heterogeneity and is also applicable to organisms like bacteria, agricultural pests, and other microbes.     

Intratumor Heterogeneity of ALK-Rearrangements and Homogeneity of EGFR-Mutations in Mixed Lung Adenocarcinoma

Background

Non Small Cell Lung Cancer is a highly heterogeneous tumor. Histologic intratumor heterogeneity could be ‘major’, characterized by a single tumor showing two different histologic types, and ‘minor’, due to at least 2 different growth patterns in the same tumor. Therefore, a morphological heterogeneity could reflect an intratumor molecular heterogeneity. To date, few data are reported in literature about molecular features of the mixed adenocarcinoma. The aim of our study was to assess EGFR-mutations and ALK-rearrangements in different intratumor subtypes and/or growth patterns in a series of mixed adenocarcinomas and adenosquamous carcinomas.

Methods

590 Non Small Cell Lung Carcinomas tumor samples were revised in order to select mixed adenocarcinomas with available tumor components. Finally, only 105 mixed adenocarcinomas and 17 adenosquamous carcinomas were included in the study for further analyses. Two TMAs were built selecting the different intratumor histotypes. ALK-rearrangements were detected through FISH and IHC, and EGFR-mutations were detected through IHC and confirmed by RT-PCR.

Results

10/122 cases were ALK-rearranged and 7 from those 10 showing an intratumor heterogeneity of the rearrangements. 12/122 cases were EGFR-mutated, uniformly expressing the EGFR-mutated protein in all histologic components.

Conclusion

Our data suggests that EGFR-mutations is generally homogeneously expressed. On the contrary, ALK-rearrangement showed an intratumor heterogeneity in both mixed adenocarcinomas and adenosquamous carcinomas. The intratumor heterogeneity of ALK-rearrangements could lead to a possible impact on the therapeutic responses and the disease outcomes.     

Intratumor heterogeneity: Nature and biological significance

Intratumor heterogeneity inherent in the majority of human cancers is a major obstacle for a highly efficient diagnosis and successful prognosis and treatment of these diseases. Being a result of clonal diversity within the same tumor, intratumor heterogeneity can be manifested in variability of genetic and epigenetic status, gene and protein expression, morphological structure, and other features of the tumor. It is most likely that the appearance of this diversity is a source for the adaptation of the tumor to changes in microenvironmental conditions and/or a tool for changing its malignant potential. In any case, both processes result in the appearance of cell clones with different undetermined sets of hallmarks. In this review, we describe the heterogeneity of molecular disorders in various human tumors and consider modern viewpoints of its development including genetic and non-genetic factors of heterogeneity origin and the role of cancer stem cells and clonal evolution. We also systematize data on the contribution of tumor diversity to progression of various tumors and the efficiency of their treatment. The main problems are indicated in the diagnosis and therapy of malignant tumors caused by intratumor heterogeneity and possible pathways for their solution. Moreover, we also suggest the key goals whose achievement promises to minimize the problem of intratumor heterogeneity and to identify new prognostic, predictive, and target markers for adequate and effective treatment of cancer.     

Proteome Heterogeneity in Colorectal Cancer

Tumor heterogeneity is an important feature of colorectal cancer (CRC) manifested by dynamic changes in gene expression, protein expression, and availability of different tumor subtypes. Recent publications in the past 10 years have revealed proteome heterogeneity between different colorectal tumors and within the same tumor site. This paper reviews recent research works on the proteome heterogeneity in CRC, which includes the heterogeneity within a single tumor (intratumor heterogeneity), between different anatomical sites at the same organ, and between primary and metastatic sites (intertumor heterogeneity). The potential use of proteome heterogeneity in precision medicine and its implications in biomarker discovery and therapeutic outcomes will be discussed. Identification of the unique proteome landscape between and within individual tumors is imperative for understanding cancer biology and the management of CRC patients.     

Intratumor heterogeneity of k-ras and p53 mutations among human colorectal adenomas containing early cancer.

The molecular pathways and the timing of genetic events during human colorectal carcinogenesis are still not fully understood. We have addressed the intratumor heterogeneity of the mutational status of the k-ras oncogene and of the p53 oncosuppressor gene during the adenoma-carcinoma sequence by investigating 26 human colorectal adenomas containing early cancer. An intratumor comparative analysis was obtained among the adenomatous and carcinomatous component pairs. Additionally, we have analyzed 17 adenomas having cancer in the near vicinity. The adenomatous components of the adenomas containing early cancer and the adenomas having cancer in the near vicinity had comparable frequencies for k-ras mutations (28 and 47%) but different for p53 mutations (52 and 7%, p-value = 0.01). Interestingly, the adenomatous and carcinomatous components of the adenomas containing early cancer were rarely heterogeneous for the k-ras mutational status (only in 13% of the cases) but were characterized by heterogeneity of the p53 status in 59% of the cases (p-value < 0.01). In addition, the mutations of p53 for the adenomatous components of the adenomas containing early cancer were statistically significantly associated with severe dysplasia (p-value = 0.01). Intratumor homogeneity of k-ras status during the human colorectal adenoma-carcinoma sequence suggests that the role of k-ras is more related to tumor initiation than to tumor progression. On the contrary, intratumor heterogeneity of p53 mutations indicates that the type of the p53 mutations may also be relevant for selection and expansion of new subclones leading to tumor progression.     

Intratumor heterogeneity of chromosome 1, 7, 17, and 18 aneusomies obtained by FISH and association with flow cytometric DNA index in human colorectal adenocarcinomas

BACKGROUND: The origin and evolution of somatic chromosome aberrations in colorectal cancer is still poorly understood. The data in the literature suggest that some specific chromosome aberrations are more common. It is not known, however, if there is a correlation of these with near-diploid and high aneuploidy previously proposed to be a characteristic of the adenoma-carcinoma sequence. METHODS: Chromosome 1, 7, 17 and 18 numerical aberrations and 1p deletions were evaluated by fluorescence in situ hybridization analysis for 20 human sporadic colorectal adenocarcinomas in 70 distinct tumor sectors and correlated with flow cytometric DNA index (DI) values. RESULTS: Aneusomy for at least one of the investigated chromosomes was observed in 60 of 70 tumor sectors corresponding to 19 of 20 adenocarcinomas (95%). Deletions at 1p, observed in 8 of 18 adenocarcinomas (44%), were intratumor homogeneous in 7 of 8 tumors. In contrast, the other aberrations were intratumor heterogeneous. Aneusomies of chromosomes 1, 7, and 17 were strongly associated with DNA high aneuploidy (DI > or = 1.4), whereas aneusomy of chromosome 18 and 1p deletions were equally common among DNA diploid and near-diploid tumors (DI < 1.4 and DI not equal to 1). CONCLUSIONS: Overall, these data suggest the existence of different aneuploidization routes correlated with specific chromosome aberrations. In addition, intratumor homogeneity of 1p deletions appears to be an indication of early occurrence or strong selection. We also suggest that tumors with monosomies and in particular monosomies-trisomies for the same chromosomes support a model of aneuploidization and chromosome instability during the colorectal tumor progression based on loss of symmetry during chromosome segregation (Giaretti: Lab Invest 71:904-910, 1994).     

Unsupervised Deconvolution of Dynamic Imaging Reveals Intratumor Vascular Heterogeneity and Repopulation Dynamics

With the existence of biologically distinctive malignant cells originated within the same tumor, intratumor functional heterogeneity is present in many cancers and is often manifested by the intermingled vascular compartments with distinct pharmacokinetics. However, intratumor vascular heterogeneity cannot be resolved directly by most in vivo dynamic imaging. We developed multi-tissue compartment modeling (MTCM), a completely unsupervised method of deconvoluting dynamic imaging series from heterogeneous tumors that can improve vascular characterization in many biological contexts. Applying MTCM to dynamic contrast-enhanced magnetic resonance imaging of breast cancers revealed characteristic intratumor vascular heterogeneity and therapeutic responses that were otherwise undetectable. MTCM is readily applicable to other dynamic imaging modalities for studying intratumor functional and phenotypic heterogeneity, together with a variety of foreseeable applications in the clinic.     

Centromere cleavage is a mechanism underlying isochromosome formation in skin and head and neck carcinomas

Centromeric rearrangements, in the form of isochromosomes or whole-arm translocations, are the most common recurrent changes in head and neck and skin carcinomas. Little is known about the mechanisms behind the origin of these chromosome rearrangements. In the present study, one basal cell carcinoma and two squamous cell carcinomas of the head and neck were thoroughly studied by cytogenetic and fluorescence in situ hybridization techniques. All tumors showed intratumor heterogeneity in the form of cytogenetically related subclones (in all tumors) and unrelated clones (in one tumor). Assessment of karyotypic evolution in these tumors suggests that centromeric cleavage is a mechanism giving rise to isochromosomes. A similar mechanism may also be involved in the formation of whole-arm translocations.     

CpG Dinucleotide Frequencies Reveal the Role of Host Methylation Capabilities in Parvovirus Evolution

Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to “fractional” methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.     

Genomic heterogeneity and instability in colorectal cancer: spectral karyotyping, glutathione transferase-Ml and ras

Genomic instability in cancer is frequently described as being either chromosomal instability or microsatellite instability, although when events within chromosomes are monitored, extensive intrachromosomal instability is also found. Spectral karyotyping was used to visualize how extensively genomic instability gives rise to intratumor genomic heterogeneity in sporadic colorectal carcinomas. Two factors were then examined which might relate to intrachromosomal instability in colorectal cancers: the presence of the glutathione transferase-Ml gene to detoxify potential carcinogens, and the presence of activated ras which has been associated with chromosomal instability when first expressed. Intrachromosomal genomic instability was previously determined by inter-(simple sequence repeat) PCR (inter-SSR PCR) and by fractional allelic loss rate for 348 markers. GSTM1 status was determined for each of 49 tumors through use of specific PCR, and 28 of the tumors showed the GSTM1 null genotype. A significant association was found between GSTMl-null status and elevated inter-(simple sequence repeat) PCR instability. In contrast, no association was found with fractional allelic loss rate. The first exons of the K-ras and H-ras oncogenes were sequenced in 72 colorectal cancers; 19 of the tumors had a mutation in codon 12 of the K-ras gene (24.5%), but no H-ras mutations were found. A weak correlation (p=0.10) was observed between mutant K-ras and inter-(simple sequence repeat) PCR genomic instability, and no association existed with fractional allelic loss rate.     

Algorithms to Model Single Gene,Single Chromosome,and Whole Genome Copy Number Changes Jointly in Tumor Phylogenetics

We present methods to construct phylogenetic models of tumor progression at the cellular level that include copy number changes at the scale of single genes, entire chromosomes, and the whole genome. The methods are designed for data collected by fluorescence in situ hybridization (FISH), an experimental technique especially well suited to characterizing intratumor heterogeneity using counts of probes to genetic regions frequently gained or lost in tumor development. Here, we develop new provably optimal methods for computing an edit distance between the copy number states of two cells given evolution by copy number changes of single probes, all probes on a chromosome, or all probes in the genome. We then apply this theory to develop a practical heuristic algorithm, implemented in publicly available software, for inferring tumor phylogenies on data from potentially hundreds of single cells by this evolutionary model. We demonstrate and validate the methods on simulated data and published FISH data from cervical cancers and breast cancers. Our computational experiments show that the new model and algorithm lead to more parsimonious trees than prior methods for single-tumor phylogenetics and to improved performance on various classification tasks, such as distinguishing primary tumors from metastases obtained from the same patient population.     

Evolutionary dynamics of intratumor heterogeneity

Intraneoplastic diversity in human tumors is a widespread phenomenon of critical importance for tumor progression and the response to therapeutic intervention. Insights into the evolutionary events that control tumor heterogeneity would be a major breakthrough in our comprehension of cancer development and could lead to more effective prevention methods and therapies. In this paper, we design an evolutionary mathematical framework to study the dynamics of heterogeneity over time. We consider specific situations arising during tumorigenesis, such as the emergence of positively selected mutations ("drivers") and the accumulation of neutral variation ("passengers"). We perform exact computer simulations of the emergence of diverse tumor cell clones over time, and derive analytical estimates for the extent of heterogeneity within a population of cancer cells. Our methods contribute to a quantitative understanding of tumor heterogeneity and the impact of heritable alterations on this tumor trait.     

Evolution of the cancer genome

Human tumors result from an evolutionary process operating on somatic cells within tissues, whereby natural selection operates on the phenotypic variability generated by the accumulation of genetic, genomic and epigenetic alterations. This somatic evolution leads to adaptations such as increased proliferative, angiogenic, and invasive phenotypes. In this review we outline how cancer genomes are beginning to be investigated from an evolutionary perspective. We describe recent progress in the cataloging of somatic genetic and genomic alterations, and investigate the contributions of germline as well as epigenetic factors to cancer genome evolution. Finally, we outline the challenges facing researchers who investigate the processes driving the evolution of the cancer genome.     

Molecular structure and evolution mechanism of two populations of double minutes in human colorectal cancer cells

Gene amplification chiefly manifests as homogeneously stained regions (HSRs) or double minutes (DMs) in cytogenetically and extrachromosomal DNA (ecDNA) in molecular genetics. Evidence suggests that gene amplification is becoming a hotspot for cancer research, which may be a new treatment strategy for cancer. DMs usually carry oncogenes or chemoresistant genes that are associated with cancer progression, occurrence and prognosis. Defining the molecular structure of DMs will facilitate understanding of the molecular mechanism of tumorigenesis. In this study, we re‐identified the origin and integral sequence of DMs in human colorectal adenocarcinoma cell line NCI‐H716 by genetic mapping and sequencing strategy, employing high‐resolution array‐based comparative genomic hybridization, high‐throughput sequencing, multiplex‐fluorescence in situ hybridization and chromosome walking techniques. We identified two distinct populations of DMs in NCI‐H716, confirming their heterogeneity in cancer cells, and managed to construct their molecular structure, which were not investigated before. Research evidence of amplicons distribution in two different populations of DMs suggested that a multi‐step evolutionary model could fit the module of DM genesis better in NCI‐H716 cell line. In conclusion, our data implicated that DMs play a very important role in cancer progression and further investigation is necessary to uncover the role of the DMs.     

Methylome profiling of cancer cells by amplification of inter-methylated sites (AIMS)

Alterations of the DNA methylation pattern have been related to generalized chromosomal disruption and inactivation of multiple tumor suppressor genes in neoplasia. To screen for tumor-specific alterations and to make a global assessment of methylation status in cancer cells, we have modified the methylated CpG island amplification method to generate easily readable fingerprints representing the cell’s DNA methylation profile. The method is based on the differential cleavage of isoschizomers with distinct methylation sensitivity. Specific adaptors are ligated to the methylated ends of the digested genomic DNA. The ligated sequences are amplified by PCR using adaptor- specific primers extended at the 3′ end with two to four arbitrarily chosen nucleotidic residues to reduce the complexity of the product. Fingerprints consist of multiple anonymous bands, representing DNA sequences flanked by two methylated sites, which can be isolated and individually characterized. Hybridization of the whole product to metaphase chromosomes revealed that most bands originate from the isochore H3, which identifies the regions of the genome with the highest content of CpG islands and genes. Comparison of the fingerprints obtained from normal colon mucosa, colorectal carcinomas and cell lines revealed tumor-specific alterations that are putative recurrent markers of the disease and include tumor-specific hypo- and hypermethylations.     

Somatic Mutations,Allele Loss,and DNA Methylation of the Cub and Sushi Multiple Domains 1 (CSMD1) Gene Reveals Association with Early Age of Diagnosis in Colorectal Cancer Patients

Background

The Cub and Sushi Multiple Domains 1 (CSMD1) gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients.

Methods

We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A.

Results

Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%). The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15∶1, a ratio significantly higher than the expected 2∶1 ratio (p = 0.014). This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%). Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years) than those without somatic mutations (average age, 68 years). The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%), suggesting extensive cytosine methylation predisposing to somatic mutations.

Conclusions

Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical presentation in colorectal tumors, thus further implicating CSMD1 as a tumor suppressor gene.     

Depletion of CpG Dinucleotides in Papillomaviruses and Polyomaviruses: A Role for Divergent Evolutionary Pressures

Background

Papillomaviruses and polyomaviruses are small ds-DNA viruses infecting a wide-range of vertebrate hosts. Evidence supporting co-evolution of the virus with the host does not fully explain the evolutionary path of papillomaviruses and polyomaviruses. Studies analyzing CpG dinucleotide frequencies in virus genomes have provided interesting insights on virus evolution. CpG dinucleotide depletion has not been extensively studied among papillomaviruses and polyomaviruses. We sought to analyze the relative abundance of dinucleotides and the relative roles of evolutionary pressures in papillomaviruses and polyomaviruses.

Methods

We studied 127 full-length sequences from papillomaviruses and 56 full-length sequences from polyomaviruses. We analyzed the relative abundance of dinucleotides, effective codon number (ENC), differences in synonymous codon usage. We examined the association, if any, between the extent of CpG dinucleotide depletion and the evolutionary lineage of the infected host. We also investigated the contribution of mutational pressure and translational selection to the evolution of papillomaviruses and polyomaviruses.

Results

All papillomaviruses and polyomaviruses are CpG depleted. Interestingly, the evolutionary lineage of the infected host determines the extent of CpG depletion among papillomaviruses and polyomaviruses. CpG dinucleotide depletion was more pronounced among papillomaviruses and polyomaviruses infecting human and other mammals as compared to those infecting birds. Our findings demonstrate that CpG depletion among papillomaviruses is linked to mutational pressure; while CpG depletion among polyomaviruses is linked to translational selection. We also present evidence that suggests methylation of CpG dinucleotides may explain, at least in part, the depletion of CpG dinucleotides among papillomaviruses but not polyomaviruses.

Conclusions

The extent of CpG depletion among papillomaviruses and polyomaviruses is linked to the evolutionary lineage of the infected host. Our results highlight the existence of divergent evolutionary pressures leading to CpG dinucleotide depletion among small ds-DNA viruses infecting vertebrate hosts.