Germline mutations and somatic inactivation of TRIM28 in Wilms tumour.

Wilms tumour is a childhood tumour that arises as a consequence of somatic and rare germline mutations, the characterisation of which has refined our understanding of nephrogenesis and carcinogenesis. Here we report that germline loss of function mutations in TRIM28 predispose children to Wilms tumour. Loss of function of this transcriptional co-repressor, which has a role in nephrogenesis, has not previously been associated with cancer. Inactivation of TRIM28, either germline or somatic, occurred through inactivating mutations, loss of heterozygosity or epigenetic silencing. TRIM28-mutated tumours had a monomorphic epithelial histology that is uncommon for Wilms tumour. Critically, these tumours were negative for TRIM28 immunohistochemical staining whereas the epithelial component in normal tissue and other Wilms tumours stained positively. These data, together with a characteristic gene expression profile, suggest that inactivation of TRIM28 provides the molecular basis for defining a previously described subtype of Wilms tumour, that has early age of onset and excellent prognosis.

The developmental programme for genesis of the entire kidney is recapitulated in Wilms tumour.

Wilms tumour (WT) is an embryonal tumour that recapitulates kidney development. The normal kidney is formed from two distinct embryological origins: the metanephric mesenchyme (MM) and the ureteric bud (UB). It is generally accepted that WT arises from precursor cells in the MM; however whether UB-equivalent structures participate in tumorigenesis is uncertain. To address the question of the involvement of UB, we assessed 55 Wilms tumours for the molecular features of MM and UB using gene expression profiling, immunohistochemsitry and immunofluorescence. Expression profiling primarily based on the Genitourinary Molecular Anatomy Project data identified molecular signatures of the UB and collecting duct as well as those of the proximal and distal tubules in the triphasic histology group. We performed immunolabeling for fetal kidneys and WTs. We focused on a central epithelial blastema pattern which is the characteristic of triphasic histology characterized by UB-like epithelial structures surrounded by MM and MM-derived epithelial structures, evoking the induction/aggregation phase of the developing kidney. The UB-like epithelial structures and surrounding MM and epithelial structures resembling early glomerular epithelium, proximal and distal tubules showed similar expression patterns to those of the developing kidney. These observations indicate WTs can arise from a precursor cell capable of generating the entire kidney, such as the cells of the intermediate mesoderm from which both the MM and UB are derived. Moreover, this provides an explanation for the variable histological features of mesenchymal to epithelial differentiation seen in WT.

Genome-wide methylation analysis identifies a core set of hypermethylated genes in CIMP-H colorectal cancer.

BACKGROUND: Aberrant DNA methylation profiles are a characteristic of all known cancer types, epitomized by the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). Hypermethylation has been observed at CpG islands throughout the genome, but it is unclear which factors determine whether an individual island becomes methylated in cancer. METHODS: DNA methylation in CRC was analysed using the Illumina HumanMethylation450K array. Differentially methylated loci were identified using Significance Analysis of Microarrays (SAM) and the Wilcoxon Signed Rank (WSR) test. Unsupervised hierarchical clustering was used to identify methylation subtypes in CRC. RESULTS: In this study we characterized the DNA methylation profiles of 94 CRC tissues and their matched normal counterparts. Consistent with previous studies, unsupervized hierarchical clustering of genome-wide methylation data identified three subtypes within the tumour samples, designated CIMP-H, CIMP-L and CIMP-N, that showed high, low and very low methylation levels, respectively. Differential methylation between normal and tumour samples was analysed at the individual CpG level, and at the gene level. The distribution of hypermethylation in CIMP-N tumours showed high inter-tumour variability and appeared to be highly stochastic in nature, whereas CIMP-H tumours exhibited consistent hypermethylation at a subset of genes, in addition to a highly variable background of hypermethylated genes. EYA4, TFPI2 and TLX1 were hypermethylated in more than 90% of all tumours examined. One-hundred thirty-two genes were hypermethylated in 100% of CIMP-H tumours studied and these were highly enriched for functions relating to skeletal system development (Bonferroni adjusted p value =2.88E-15), segment specification (adjusted p value =9.62E-11), embryonic development (adjusted p value =1.52E-04), mesoderm development (adjusted p value =1.14E-20), and ectoderm development (adjusted p value =7.94E-16). CONCLUSIONS: Our genome-wide characterization of DNA methylation in colorectal cancer has identified 132 genes hypermethylated in 100% of CIMP-H samples. Three genes, EYA4, TLX1 and TFPI2 are hypermethylated in >90% of all tumour samples, regardless of CIMP subtype.

Inflammatory and regulatory T cells contribute to a unique immune microenvironment in tumor tissue of colorectal cancer patients.

Colorectal cancer is one of the five leading causes of cancer mortality worldwide. The mechanisms of pathogen clearance, inflammation and regulation by T cells in the healthy bowel are also important in controlling tumor growth. The majority of studies analyzing T cells and their relationship to colorectal tumor growth have focused on individual T cell markers or gene clusters and thus the complexity of the T cell response contributing to the growth of the tumor is not clear. We have studied the T cells in colorectal cancer patients and have defined a unique T cell signature for colorectal tumor tissue. Using a novel analytical flow cytometric approach in concert with confocal microscopy, we have shown that the tumor has a lower frequency of effector T cells (CD69+), but a higher frequency of both regulatory (CD25hi Foxp3+) and inflammatory T cells (IL-17+) compared with associated nontransformed bowel tissue. We have also identified minor populations of T cells expressing conventional markers of both inflammatory and regulatory T cells (CD4+IL-17+Foxp3+) in the tumor tissue. These cells may represent intermediate populations or they may dictate an inflammatory versus regulatory function in surrounding T cells. Together, these data describe an immune microenvironment in colorectal cancer unique to the tumor tissue and distinct from the surrounding healthy bowel tissue, and this distinct environment is reflected by a gradient of T cells expressing markers of multiple T cell populations. These findings may be used to improve diagnosis and prognosis of colorectal cancer patients.

Global demethylation in loss of imprinting subtype of Wilms tumor.

Epigenetic abnormalities at the IGF2/H19 locus play a key role in the onset of Wilms tumor. These tumors can be classified into three molecular subtypes depending on the events occurring at this locus: loss of imprinting (LOI), loss of heterozygosity (LOH), or retention of imprinting (ROI). As IGF2 LOI is a consequence of aberrant methylation, we hypothesized that this subtype of Wilms tumors might display global abnormalities of methylation. We therefore analyzed the methylation status of satellite DNA, as a surrogate for global methylation in 50 Wilms tumor patients. Satellite methylation was quantified by a methylation-sensitive quantitative PCR. We confirmed hypomethylation of both satellite α (Sat α) and satellite 2 (Sat 2) DNA in Wilms tumor samples compared with normal kidney. In addition, we found that LOI tumors, unlike ROI or LOH ones, showed concordant hypomethylation of both Sat α and Sat 2 DNA. This would suggest that the LOI subtype of Wilms tumor, which unlike other subtypes results from an epimutation, has a global deregulation of methylation mechanisms.

T cell subpopulations in lymph nodes may not be predictive of patient outcome in colorectal cancer.

BACKGROUND: The immune response has been proposed to be an important factor in determining patient outcome in colorectal cancer (CRC). Previous studies have concentrated on characterizing T cell populations in the primary tumour where T cells with regulatory effect (Foxp3+ Tregs) have been identified as both enhancing and diminishing anti-tumour immune responses. No previous studies have characterized the T cell response in the regional lymph nodes in CRC. METHODS: Immunohistochemistry was used to analyse CD4, CD8 or Foxp3+ T cell populations in the regional lymph nodes of patients with stage II CRC (n = 31), with (n = 13) or without (n = 18) cancer recurrence after 5 years of follow up, to determine if the priming environment for anti-tumour immunity was associated with clinical outcome. RESULTS: The proportions of CD4, CD8 or Foxp3+ cells in the lymph nodes varied widely between and within patients, and there was no association between T cell populations and cancer recurrence or other clinicopathological characteristics. CONCLUSIONS: These data indicate that frequency of these T cell subsets in lymph nodes may not be a useful tool for predicting patient outcome.

WTX mutations can occur both early and late in the pathogenesis of Wilms tumour.

BACKGROUND: Somatic mutations in the X-linked tumour suppressor gene WTX have been observed in 6- 30% of sporadic cases of Wilms tumour. Germline mutations in the same gene cause the sclerosing skeletal dysplasia, osteopathia striata congenita with cranial sclerosis (OSCS). No evidence points towards a susceptibility to the development of tumours in individuals with OSCS, suggesting that there are unrecognised additional determinants that influence the phenotypic outcome associated with germline mutations in WTX. One explanation may be that a somatic mutation in WTX may need to occur late in tumour development to contribute to tumourigenesis. METHODS: Here a panel of four sporadic Wilms tumours with associated nephrogenic rest tissue and characterised WTX and CTNNB1 mutations is studied to ascertain the temporal sequence of acquisition of these mutations. Additionally, a family with OSCS is described segregating a germline mutation in WTX and manifesting a lethal phenotype in males. One male from this family had bilateral multifocal nephrogenic rests at autopsy. RESULTS: In one of the four tumours the WTX mutation was present in both tumour and rest tissue indicating it had arisen early in tumour development. In the remaining three tumours, the WTX mutation was present in the tumour only indicating late acquisition of these mutations. CONCLUSIONS: These data indicate that WTX mutations can arise both early and late in Wilms tumour development. WTX mutations may predispose to nephrogenic rest development rather than Wilms tumour per se.

Gene expression profiles as predictors of poor outcomes in stage II colorectal cancer: A systematic review and meta-analysis.

PURPOSE: The use of adjuvant therapy in stage II colorectal cancer (CRC) remains controversial. There is a need to identify more effective predictors than the traditional staging system to aid therapeutic decision-making. We performed a systematic review and meta-analysis of gene expression profiles (GEPs) to assess their utility for risk stratification and prediction of poor outcomes in stage II CRC. PATIENTS AND METHODS: We performed a comprehensive literature search through December 2007. Studies were included if they reported GEP-based assays in patients with stage II CRC, and either subsequent cancer recurrence or death within 3 years. The prognostic likelihood ratio (LR) and odds ratio (OR) were calculated with 95% confidence intervals and pooled using the fixed-effects method. The weighted average sensitivity, specificity, and accuracy were also reported. RESULTS: Eight cohorts involving 271 patients contributed to the analysis. The average accuracy, sensitivity, and specificity were 81.9%, 76.2%, and 84.5%, respectively, with a prognostic LR of 4.7 (95% CI, 3.2-6.8) and a prognostic OR of 15.1 (95% CI, 7.9-28.6). No evidence for significant interstudy heterogeneity was noted in either analysis. Subgroup analysis found no difference in results for the prediction of cancer recurrence or death. CONCLUSION: This analysis demonstrates the promising potential of using GEP assays as predictors of poor outcomes in stage II CRC, such as cancer recurrence or death. To maximize their utility and availability, further studies will be needed to identify and validate specific gene signatures for poor prognosis in stage II CRC.

PAX3 is expressed in the stromal compartment of the developing kidney and in Wilms tumors with myogenic phenotype.

Wilms tumor (WT) is the most frequent renal neoplasm of childhood; a myogenic component is observed in 5% to 10% of tumors. We demonstrate for the first time that myogenic WTs are associated with expression of PAX3, a transcription factor known to specify myoblast cell fate during muscle development. In a panel of 20 WTs, PAX3 was identified in 13 of 13 tumor samples with myogenic histopathology but was absent in 7 of 7 tumors lacking a myogenic component. Furthermore, we show that PAX3 is expressed in the metanephric mesenchyme and stromal compartment of developing mouse kidney. Modulation of endogenous PAX3 expression in human embryonic kidney (HEK293) cells influenced cell migration in in vitro assays. Mutations of WT1 were consistently associated with PAX3 expression in WTs, and modulation of WT1 expression in HEK293 cells was inversely correlated with the level of endogenous PAX3 protein. We demonstrate abundant PAX3 and absence of PAX2 expression in a novel cell line (WitP3) isolated from the stromal portion of a WT bearing a homozygous deletion of the WT1 gene. We hypothesize that PAX3 sets stromal cell fate in developing kidney but is normally suppressed by WT1 during the mesenchyme-to-epithelium transition leading to nephrogenesis. Loss of WT1 permits aberrant PAX3 expression in a subset of WTs with myogenic phenotype.

Heterogeneous gene expression changes in colorectal cancer cells share the WNT pathway in response to growth suppression by APHS-mediated COX-2 inhibition.

Cyclooxygenase-2 (COX-2), the prostaglandin (PG)-synthesizing enzyme overexpressed in colorectal cancer (CRC), has pleiotropic, cancer-promoting effects. COX-2 inhibitors (CIBs) interfere with many cancer-associated processes and show promising antineoplastic activity, however, a common mechanism of CIB action has not yet been established. We therefore investigated by microarray the global response towards the CIB APHS at a dose significantly inhibiting the growth of three COX-2-positive CRC but not of two COX-2-negative cell lines. None of the genes significantly (p = 0.005) affected by APHS were common to all three cell lines and 83% of the altered pathways were cell line-specific. Quantitative polymerase chain reaction (QPCR) on selected pathways confirmed cell line-specific expression alterations induced by APHS. A low stringency data analysis approach using BRB array tools coupled with QPCR, however, identified small expression changes shared by all COX-2-positive cell lines in genes related to the WNT pathway, the key driver of colonic carcinogenesis. Our data indicates a substantial cell line-specificity of APHS-induced expression alterations in CRC cells and helps to explain the divergent effects reported for CIBs. Further, the shared inhibition of the WNT pathway by APHS suggests one potential common mechanism behind the antineoplastic effects of COX-2 inhibition.

Molecular pathology and epidemiology of nephrogenic rests and Wilms tumors.

Perilobar (PLNR) and intralobar nephrogenic rests (ILNR) are distinct precursor lesions of Wilms tumors that have different structural, clinical, genetic, and epidemiologic features. Wilms tumors in East-Asian children have unique epidemiologic features in that the incidence is about half that of white children, an early age at diagnosis, a male predominance, and an association with ILNR. Loss of IGF2 imprinting is associated with PLNR more commonly seen in Wilms tumors from white children than tumors from children of Asian descent. Therefore, this epigenetic difference and the higher frequency of PLNR provide an explanation for the interethnic variations in the incidence of Wilms tumor. The histopathologic, clinical, and genetic differences between ILNR and PLNR are described in this review, followed by a description of an epigenetic mechanism that underlies PLNR formation and the unique epidemiologic feature of Wilms tumors.

Destabilized adhesion in the gastric proliferative zone and c-Src kinase activation mark the development of early diffuse gastric cancer.

The initial development of diffuse gastric cancer (DGC) is poorly understood. The study of E-cadherin (CDH1) germ line mutation carriers predisposed to DGC provides a rare opportunity to elucidate the genetic and biological events surrounding disease initiation. Samples from various stages of hereditary and sporadic DGC were investigated to determine general mechanisms underlying early DGC development. Paraffin-embedded tissues from 13 CDH1 mutation carriers and from 10 sporadic early DGC cases were analyzed. Immunofluorescence and immunohistochemistry using differentiation, proliferation, and adhesion markers showed that DGC initiation seems to occur at the proliferative zone (the upper neck) of the gastric epithelium and correlates with absent or reduced expression of junctional proteins (beta-actin, p120, Lin-7). Slow proliferation of neoplastic cells at the upper gastric neck leads to the formation of intramucosal signet-ring cell carcinoma (SRCC) displaying differentiated features. As shown by immunolabeling, invasion from SRCC lesions beyond the gastric mucosa is associated with poor differentiation, increased proliferation, activation of the c-Src system, and an epithelial-mesenchymal transition. Our results provide a molecular description of the early development of DGC and explain the relationship between the two main DGC types, poorly differentiated carcinoma and SRCC: both share their origin, but SRCC develops following cancer cell differentiation and seems relatively indolent in its intramucosal stage.

Multiple gene expression classifiers from different array platforms predict poor prognosis of colorectal cancer.

PURPOSE: This study aimed to develop gene classifiers to predict colorectal cancer recurrence. We investigated whether gene classifiers derived from two tumor series using different array platforms could be independently validated by application to the alternate series of patients. EXPERIMENTAL DESIGN: Colorectal tumors from New Zealand (n = 149) and Germany (n = 55) patients had a minimum follow-up of 5 years. RNA was profiled using oligonucleotide printed microarrays (New Zealand samples) and Affymetrix arrays (German samples). Classifiers based on clinical data, gene expression data, and a combination of the two were produced and used to predict recurrence. The use of gene expression information was found to improve the predictive ability in both data sets. The New Zealand and German gene classifiers were cross-validated on the German and New Zealand data sets, respectively, to validate their predictive power. Survival analyses were done to evaluate the ability of the classifiers to predict patient survival. RESULTS: The prediction rates for the New Zealand and German gene-based classifiers were 77% and 84%, respectively. Despite significant differences in study design and technologies used, both classifiers retained prognostic power when applied to the alternate series of patients. Survival analyses showed that both classifiers gave a better stratification of patients than the traditional clinical staging. One classifier contained genes associated with cancer progression, whereas the other had a large immune response gene cluster concordant with the role of a host immune response in modulating colorectal cancer outcome. CONCLUSIONS: The successful reciprocal validation of gene-based classifiers on different patient cohorts and technology platforms supports the power of microarray technology for individualized outcome prediction of colorectal cancer patients. Furthermore, many of the genes identified have known biological functions congruent with the predicted outcomes.

Sequential WT1 and CTNNB1 mutations and alterations of beta-catenin localisation in intralobar nephrogenic rests and associated Wilms tumours: two case studies.

BACKGROUND: Intralobar nephrogenic rests (ILNRs) are precursor lesions for Wilms tumours and are associated with WT1 gene mutations. ILNR-associated Wilms tumours have a co-clustering of WT1 and beta-catenin (CTNNB1) mutations and unique histological features characterised by a stromal-predominant histology. AIM: To determine the order in which WT1 and CTNNB1 mutations occur to understand the ILNR-Wilms tumour sequence. METHODS: Of nine Wilms tumours with WT1 and CTNNB1 mutations, three ILNRs lesions in two Wilms tumours were available for analysis of WT1 and CTNNB1 mutations using microdissection. Immunohistochemistry was also performed to investigate how the mutations in beta-catenin alter the localisation in Wilms tumour development. RESULTS: WT1 mutations were present in the ILNRs, however CTNNB1 mutations were absent. Immunohistochemistry for WT1 confirmed inactivation of WT1 in both ILNRs and Wilms tumours. Both the ILNRs and the associated Wilms tumours had similar immunostaining patterns for beta-catenin in the blastemal and epithelial components. Although rhabdomyoblasts were not included in ILNRs, the associated Wilms tumours showed rhabdomyogenic differentiation with a positive beta-catenin nuclear staining. CONCLUSIONS: The results suggest that CTNNB1 mutation is a later event in Wilms tumourigenesis. CTNNB1 mutations might be associated with rhabdomyogenesis.

Age distributions, birth weights, nephrogenic rests, and heterogeneity in the pathogenesis of Wilms tumor.

BACKGROUND: The National Wilms Tumor Study (NWTS) constitutes a unique resource for study of clinical, pathologic, and epidemiologic features of Wilms tumor (WT). PROCEDURE: Data from NWTS-3,4,5 were compiled for 7,455 patients with tumors of favorable (FH) or anaplastic (AH) histology. The associations of birth weight (BW) and age-at-onset with gender, intralobar (ILNR), and perilobar (PLNR) nephrogenic rests, tumor focality, congenital malformation syndromes, and tumor histology were analyzed using descriptive statistics and linear regression. RESULTS: Mean BWs for male and female patients without PLNR were 3.52 and 3.36 kg, respectively, and for those with PLNR were 0.12 kg and 0.15 kg heavier. Mean age was 45 months for males with no rests whose tumors were unifocal and of triphasic favorable histology. ILNR or multifocality decreased the mean age by 18 and 10 months, respectively, whereas female gender, blastemal/FH or AH increased it by 3, 10, and 16 months. Over 90% of multifocal tumors occurred in the presence of demonstrated ILNR or PLNR or both. The apparent bimodality of the age distributions and later mean ages-at-onset for females with both unifocal and multifocal tumors were explained in part by the relative deficit in females of ILNR versus PLNR-associated tumors. CONCLUSIONS: These observations support the view that there are multiple pathways to Wilms tumorigenesis. They will facilitate selection of informative subgroups of patients for molecular analysis that may serve to identify the putative pathway for the majority of patients who cannot be classified provisionally on the basis of ILNR or PLNR.

A gene expression signature for relapse of primary wilms tumors.

Anaplastic histology and metastasis are each associated with higher relapse and mortality rates in Wilms tumor patients. However, not all anaplastic tumors relapse and some nonanaplastic tumors relapse unexpectedly. To identify more accurate early prognostic indicators, we analyzed expression of 4,900 cancer-related genes in 26 primary Wilms tumors. This analysis revealed that expression of a set of four genes predicts future relapse of primary Wilms tumors with high accuracy, independent of anaplasia. Random permutation testing of this prognostic gene expression signature yielded P = 0.003. Real-time reverse transcription-PCR analysis of the four genes in an independent primary tumor set resulted in correct prediction of future relapse with an accuracy of 92%. One of the four genes in the prognostic signature, CCAAT/enhancer binding protein beta (C/EBPB), is expressed at higher levels in both primary relapsing tumors and metastatic tumors than in primary nonrelapsing tumors. Short interfering RNA-mediated down-regulation of C/EBPB expression in WiT49, a cell line derived from a metastatic Wilms tumor, resulted in spontaneous apoptosis. These findings suggest that C/EBPB is a critical survival factor for Wilms tumor cells and that its expression contributes to the prognosis of Wilms tumor patients.

Characterization of loss-of-inactive X in Klinefelter syndrome and female-derived cancer cells.

The increased risk of several types of cancer in Klinefelter syndrome (47XXY) suggests that the extra X chromosome may be involved in the tumorigenesis associated with this syndrome. Here, we show that cancer cells (PSK-1) derived from a patient with Klinefelter syndrome (47XXY) showing loss of an inactive X chromosome subsequently gained active X chromosomes. We found that this abnormal X chromosome composition in PSK-1 is caused by a loss of an inactive X chromosome followed by multiplication of identical active X chromosomes, not by reactivation of an inactive X chromosome. Furthermore, we extended the characterization of loss-of-inactive X in a series of 22 female-derived cancer cell lines (eight breast cancer cell lines, seven ovarian cancer cell lines, and seven cervical cancer cell lines). The data demonstrate that loss-of-inactive X in the female-derived cancer cells is mainly achieved by loss of an inactive X chromosomes followed by multiplication of an identical active X chromosomes. However, distinctive pathways, including reactivation of an inactive X chromosome, are also involved in the mechanisms for loss-of-inactive X and gain-of-active X in female-derived cancer cells. The biological significance of the loss-of-inactive X and gain-of-active X in the oncogenesis of Klinefelter syndrome and female-derived cancer cells are discussed.

Myogenesis in Wilms' tumors is associated with mutations of the WT1 gene and activation of Bcl-2 and the Wnt signaling pathway.

Wilms tumors with WT1 mutations [ WT1(-)] have a stromal-predominant histology with varying extents of rhabdomyogenesis. These tumors also frequently have mutations in the beta-catenin gene ( CTNNB1). We have investigated the molecular events that may explain the origins of rhabdomyogenesis in WT1(-) tumors. Of 35 Wilms tumors, we identified 12 with WT1 mutations, of which 9 carried CTNNB1 mutations. We compared WT1 wild-type tumors [ WT1(+)] with WT1(-) tumors for histological features, localization of beta-catenin, Bcl-2 expression, and apoptosis using an in-situ end-labeling technique. WT1(+) tumors showed triphasic and blastemal- and epithelial predominant-histology. Expression of WT1, beta-catenin, and Bcl-2 recapitulated those of normal kidney epithelial development. Localization of beta-catenin was observed in the cytoplasm and cytoplasmic membrane of early glomerular epithelial structures. Bcl-2 is also expressed in condensing blastema and early glomerular epithelial structures which had little apoptosis. WT1(-) tumors, regardless of whether CTNNB1 mutations were detected or not, showed a stromal-rich phenotype with abundant expression of beta-catenin in the nucleus of the rhabdomyoblasts. Bcl-2 was expressed in rhabdomyoblasts, but not in blastemal cells undergoing apoptosis, suggesting that WT1 regulates Bcl-2 positively in the epithelial pathway, but negatively in the myogenic pathway. These data indicate that mutations in WT1 might alter the Wnt signaling pathway and Bcl-2 related-apoptosis. In WT1(-) tumors, the nuclear accumulation of beta-catenin and Bcl-2 expression are associated with rhabdomyogenesis, and dysregulation of Bcl-2 may be a mechanism by which the histogenesis (loss of blastemal component, muscle differentiation) may be explained.