The malignant transformation of precancerous colorectal lesions involves progressive alterations at both the molecular and morphologic levels, the latter consisting of increases in size and in the degree of cellular atypia. Analyzing preinvasive tumors of different sizes can therefore shed light on the sequence of these alterations.
We used a molecular pathway-based approach to analyze transcriptomic profiles of 59 colorectal tumors representing early and late preinvasive stages and the invasive stage of tumorigenesis. Random set analysis was used to identify biological pathways enriched for genes differentially regulated in tumors (compared with 59 samples of normal mucosa).
Of the 880 canonical pathways we investigated, 112 displayed significant tumor-related upregulation or downregulation at one or more stages of tumorigenesis. This allowed us to distinguish between pathways whose dysregulation is probably necessary throughout tumorigenesis and those whose involvement specifically drives progression from one stage to the next. We were also able to pinpoint specific changes within each gene set that seem to play key roles at each transition. The early preinvasive stage was characterized by cell-cycle checkpoint activation triggered by DNA replication stress and dramatic downregulation of basic transmembrane signaling processes that maintain epithelial/stromal homeostasis in the normal mucosa. In late preinvasive lesions, there was also downregulation of signal transduction pathways (e.g., those mediated by G proteins and nuclear hormone receptors) involved in cell differentiation and upregulation of pathways governing nuclear envelope dynamics and the G2>M transition in the cell cycle. The main features of the invasive stage were activation of the G1>S transition in the cell cycle, upregulated expression of tumor-promoting microenvironmental factors, and profound dysregulation of metabolic pathways (e.g., increased aerobic glycolysis, downregulation of pathways that metabolize drugs and xenobiotics).
Our analysis revealed specific pathways whose dysregulation might play a role in each transition of the transformation process. This is the first study in which such an approach has been used to gain further insights into colorectal tumorigenesis. Therefore, these data provide a launchpad for further exploration of the molecular characterization of colorectal tumorigenesis using systems biology approaches.
Colorectal adenoma; Colorectal cancer; Transcriptomics; Molecular pathways; Cell cycle pathways; Random set method
Alterations in the gene expression profile in epithelial cells during breast ductal carcinoma (DC) progression have been shown to occur mainly between pure ductal carcinoma in situ (DCIS) to the in situ component of a lesion with coexisting invasive ductal carcinoma (DCIS-IDC) implying that the molecular program for invasion is already established in the preinvasive lesion. For assessing early molecular alterations in epithelial cells that trigger tumorigenesis and testing them as prognostic markers for breast ductal carcinoma progression, we analyzed, by reverse transcription-quantitative polymerase chain reaction, eight genes previously identified as differentially expressed between epithelial tumor cells populations captured from preinvasive lesions with distinct malignant potential, pure DCIS and the in situ component of DCIS-IDC. ANAPC13 and CLTCL1 down-regulation revealed to be early events of DC progression that anticipated the invasiveness manifestation. Further down-regulation of ANAPC13 also occurred after invasion appearance and the presence of the protein in invasive tumor samples was associated with higher rates of overall and disease-free survival in breast cancer patients. Furthermore, tumors with low levels of ANAPC13 displayed increased copy number alterations, with significant gains at 1q (1q23.1–1q32.1), 8q, and 17q (17q24.2), regions that display common imbalances in breast tumors, suggesting that down-regulation of ANAPC13 contributes to genomic instability in this disease.
The natural history of bronchial preinvasive lesions and the risk of developing lung cancer in patients with these lesions are not clear. Previous studies have treated severe dysplasia and carcinoma in situ (CIS) on the assumption that most will progress to invasive carcinoma.
To define the natural history of preinvasive lesions and assess lung cancer risk in patients with these lesions.
Most preinvasive lesions will not progress to invasive carcinoma but patients with these lesions will be at high risk.
A cohort of patients with preinvasive lesions underwent fluorescence bronchoscopy every 4–12 months and computed tomography of the chest annually. The main end point was the development of invasive carcinoma.
22 patients with 53 lesions were followed up for 12–85 months. 11 cancers were diagnosed in 9 patients. Of the 36 high‐grade lesions (severe dysplasia and CIS), 6 progressed to invasive cancers. 5 separate cancers developed at remote sites in patients with high‐grade lesions. All cancers were N0M0 and curative treatment was given to 8 of the 9 patients. The cumulative risk of developing lung cancer in a patient with a high‐grade lesion was 33% and 54% at 1 and 2 years, respectively. Of the 17 low‐grade lesions, none progressed to invasive carcinoma.
Although the risk of malignant progression of individual preinvasive lesions is relatively small, patients with high‐grade lesions are at high risk of lung cancer. Surveillance facilitated early detection and treatment with curative intent in most patients.
Genomic and transcriptomic alterations affecting key cellular processes such us cell proliferation, differentiation and genomic stability are considered crucial for the development and progression of cancer. Most invasive breast carcinomas are known to derive from precursor in situ lesions. It is proposed that major global expression abnormalities occur in the transition from normal to premalignant stages and further progression to invasive stages. Serial analysis of gene expression (SAGE) was employed to generate a comprehensive global gene expression profile of the major changes occurring during breast cancer malignant evolution.
In the present study we combined various normal and tumor SAGE libraries available in the public domain with sets of breast cancer SAGE libraries recently generated and sequenced in our laboratory. A recently developed modified t test was used to detect the genes differentially expressed.
We accumulated a total of approximately 1.7 million breast tissue-specific SAGE tags and monitored the behavior of more than 25,157 genes during early breast carcinogenesis. We detected 52 transcripts commonly deregulated across the board when comparing normal tissue with ductal carcinoma in situ, and 149 transcripts when comparing ductal carcinoma in situ with invasive ductal carcinoma (P < 0.01).
A major novelty of our study was the use of a statistical method that correctly accounts for the intra-SAGE and inter-SAGE library sources of variation. The most useful result of applying this modified t statistics beta binomial test is the identification of genes and gene families commonly deregulated across samples within each specific stage in the transition from normal to preinvasive and invasive stages of breast cancer development. Most of the gene expression abnormalities detected at the in situ stage were related to specific genes in charge of regulating the proper homeostasis between cell death and cell proliferation. The comparison of in situ lesions with fully invasive lesions, a much more heterogeneous group, clearly identified as the most importantly deregulated group of transcripts those encoding for various families of proteins in charge of extracellular matrix remodeling, invasion and cell motility functions.
breast cancer; gene expression profiling; serial analysis of gene expression
Similar to the adenoma-carcinoma sequence of colorectal cancer, lung adenocarcinoma is thought to follow a linear multistep progression, in which a precursor lesion progresses to adenocarcinoma in situ, which is followed by invasive adenocarcinoma. However, lung adenocarcinoma can no longer be considered as a single type of tumor but rather a group of distinct subsets of tumors that arise from different molecular pathways. Consistent with this concept, recent findings revealed that this linear progression might not occur in all lung adenocarcinomas. First, according to the molecular classification based on expression profiling, lung cancer can be divided into at least two subsets; precancerous and in-situ lesions share characteristics of molecular expression and clinical features with only one of the two subsets, suggesting that the linear progression is only applicable to the subset in the molecular classification. Second, when EGFR and KRAS were examined based on the progression steps, the mutation rate of KRAS was disproportionally distributed; however, according to the progression schema, gene alterations should be evenly accumulated along the entire progression. Third, by means of comparative genomic hybridization analysis, some adenocarcinoma in situ revealed gene alterations discontinuous to invasive adenocarcinoma. Finally, there were some clinical observations that support that some lesions retain the progression. In this review, we hypothesize a novel scenario for the progression of lung adenocarcinoma, which does not support a linear progression schema.
precancerous lesion; carcinoma in situ; cancer progression; lung cancer; adenocarcinoma
Preinvasive bronchial lesions defined as dysplasia and carcinoma in situ (CIS) have been considered as precursors of squamous cell carcinoma of the lung. The risk and rate of progression of preinvasive lesions to invasive squamous cell carcinoma as well as the mechanism of progression or regression are incompletely understood. While the evidence for the multistage, stepwise progression model is weak with relatively few documented lesions that progress through various grades of dysplasia to CIS and then to invasive carcinoma, the concept of field carcinogenesis is strongly supported. The presence of high-grade dysplasia or CIS is a risk marker for lung cancer both in the central airways and peripheral lung. Genetic alterations such as loss of heterozygosity in chromosome 3p or chromosomal aneusomy as well as host factors such as the inflammatory load and levels of anti-inflammatory proteins in the lung influence the progression or regression of preinvasive lesions. CIS is different than severe dysplasia at the molecular level and has different clinical outcome. Molecular analysis of dysplastic lesions that progress to CIS or invasive cancer and rare lesions that progress rapidly from hyperplasia or metaplasia to CIS or invasive cancer will shed light on the key molecular determinants driving development to an invasive phenotype versus those associated with tobacco smoke damage.
Preinvasive lesions; Natural history; Lung cancer
Ductal carcinoma in situ (DCIS) of the breast includes a heterogeneous group of preinvasive tumors with uncertain evolution. Definition of the molecular factors necessary for progression to invasive disease is crucial to determining which lesions are likely to become invasive. To obtain insight into the molecular basis of DCIS, we compared the gene expression pattern of cells from the following samples: non-neoplastic, pure DCIS, in situ component of lesions with co-existing invasive ductal carcinoma, and invasive ductal carcinoma.
Forty-one samples were evaluated: four non-neoplastic, five pure DCIS, 22 in situ component of lesions with co-existing invasive ductal carcinoma, and 10 invasive ductal carcinoma. Pure cell populations were isolated using laser microdissection. Total RNA was purified, DNase treated, and amplified using the T7-based method. Microarray analysis was conducted using a customized cDNA platform. The concept of molecular divergence was applied to classify the sample groups using analysis of variance followed by Tukey's test.
Among the tumor sample groups, cells from pure DCIS exhibited the most divergent molecular profile, consequently identifying cells from in situ component of lesions with co-existing invasive ductal carcinoma as very similar to cells from invasive lesions. Additionally, we identified 147 genes that were differentially expressed between pure DCIS and in situ component of lesions with co-existing invasive ductal carcinoma, which can discriminate samples representative of in situ component of lesions with co-existing invasive ductal carcinoma from 60% of pure DCIS samples. A gene subset was evaluated using quantitative RT-PCR, which confirmed differential expression for 62.5% and 60.0% of them using initial and partial independent sample groups, respectively. Among these genes, LOX and SULF-1 exhibited features that identify them as potential participants in the malignant process of DCIS.
We identified new genes that are potentially involved in the malignant transformation of DCIS, and our findings strongly suggest that cells from the in situ component of lesions with co-existing invasive ductal carcinoma exhibit molecular alterations that enable them to invade the surrounding tissue before morphological changes in the lesion become apparent.
Background: The potential of autofluorescence bronchoscopy (AFB) to detect precancerous lesions in the central airways and its role in lung cancer screening is uncertain. A study was undertaken to evaluate the prevalence of moderate/severe dysplasia (dysplasia II–III) and carcinoma in situ (CIS) using a newly developed AFB system in comparison with conventional white light bronchoscopy (WLB) alone.
Methods: In a prospective randomised multicentre trial, smokers ⩾40 years of age (⩾20 pack-years) were stratified into four different risk groups and investigated with either WLB+AFB (arm A) or WLB alone (arm B).
Results: 1173 patients (916 men) of mean age 58.7 years were included. Overall (arms A and B), preinvasive lesions (dysplasia II–III and CIS) were detected in 3.9% of the patients. The prevalence of patients with preinvasive lesions in the WLB arm was 2.7% compared with 5.1% in the WLB+AFB arm (p = 0.037). For patients with dysplasia II–III, WLB+AFB increased the detection rate by a factor of 2.1 (p = 0.03), while for CIS the factor was only 1.24 (p = 0.75). The biopsy based sensitivity of WLB alone and WLB+AFB for detecting dysplasia II–III and CIS was 57.9% compared with 82.3% (1.42-fold increase). The corresponding specificity was 62.1% compared with 58.4% (0.94-fold decrease).
Conclusions: This first randomised study of AFB showed that the combination of WLB+AFB was significantly superior to WLB alone in detecting preneoplastic lesions. Our findings do not support the general use of AFB as a screening tool for lung cancer, but suggest that it may be of use in certain groups. The precise indications await further study.
Serial Analysis of Gene Expression (SAGE) is a new technique that allows a detailed and profound quantitative and qualitative knowledge of gene expression profile, without previous knowledge of sequence of analyzed genes. We carried out a modification of SAGE methodology (microSAGE), useful for the analysis of limited quantities of tissue samples, on normal human cervical tissue obtained from a donor without histopathological lesions. Cervical epithelium is constituted mainly by cervical keratinocytes which are the targets of human papilloma virus (HPV), where persistent HPV infection of cervical epithelium is associated with an increase risk for developing cervical carcinomas (CC).
We report here a transcriptome analysis of cervical tissue by SAGE, derived from 30,418 sequenced tags that provide a wealth of information about the gene products involved in normal cervical epithelium physiology, as well as genes not previously found in uterine cervix tissue involved in the process of epidermal differentiation.
This first comprehensive and profound analysis of uterine cervix transcriptome, should be useful for the identification of genes involved in normal cervix uterine function, and candidate genes associated with cervical carcinoma.
The progression from preinvasive lesion to invasive carcinoma is a critical step contributing to breast cancer lethality. We identified down-regulation of milk fat globule-EGF factor 8 (MFG-E8) as a contributor to breast cancer progression using microarray analysis of laser capture microdissected (LCM) tissues. We first identified MFG-E8 down-regulation in invasive lesions in transgenic mammary tumor models, which were confirmed in LCM-isolated human invasive ductal carcinomas compared with patient-matched normal tissues. In situ analyses of MFG-E8 expression in estrogen receptor (ER) positive cases confirmed its down-regulation during breast cancer progression and small inhibitory MFG-E8 RNAs accelerated ER+ breast cancer cell proliferation. MFG-E8 also decreased in erbB2+ human cancers and erbB2 transgenic mice lacking MFG-E8 showed accelerated tumor formation. In contrast, MFG-E8 expression was present at high levels in triple negative (ER-, PgR-, erbB2-) breast cancers, cell lines and patient sera. Knockdown, ChIP and reporter assays all showed that p63 regulates MFG-E8 expression, and MFG-E8 knockdowns sensitized triple negative breast cancers to cisplatin treatment. Taken together, our results show that MFG-E8 is expressed in triple negative breast cancers as a target gene of the p63 pathway, but may serve a suppressive function in ER+ and erbB2+ breast cancers. Its potential use as a serum biomarker that contributes to the pathogenesis of triple negative breast cancers urges continued evaluation of its differential functions.
cyclin D1; apoptosis; p63; integrin alpha v; integrin beta 5
Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with a poor prognosis. Cancer-associated fibroblasts (CAFs) affect tumorigenesis by creating an environment primed for growth and invasion through the secretion of factors, including hepatocyte growth factor (HGF) and transforming growth factor β1 (TGFβ1). In the present study, the levels of α-smooth muscle actin (α-SMA), TGFβ1 and HGF were determined immunohistochemically in oesophageal precancerous lesions (low- and high-grade intraepithelial neoplasia; LGIEN and HGIEN, respectively), carcinoma in situ (CIS) and squamous cell carcinoma (SCC). Immunoreactivity was observed in the cytoplasm of oesophageal epithelial cells and stromal fibroblasts. Expression levels of α-SMA, TGFβ1 and HGF increased significantly in the following order: normal, LGIEN, HGIEN, CIS and SCC. In addition, linear correlations between the expression of α-SMA, TGFβ1 and HGF and different lesions were observed. Microvessel density (MVD) was measured in all specimens and increased gradually in the normal, LGIEN, HGIEN, CIS and SCC specimens, successively. A linear correlation between MVD and pathological grade was also observed and the MVD in α-SMA-, HGF- and TGFβ1-positive groups was higher when compared with that of their negative counterparts. The results of the present study indicated that the frequent overexpression of TGFβ1 and HGF proteins, secreted by oesophageal epithelium and stromal fibroblasts, promoted the progression of oesophageal precancerous lesions via the proliferation of epithelial cells and angiogenesis, through the upregulation of vascular endothelial growth factor (VEGF) expression.
esophageal squamous cell carcinoma; fibroblast; transforming growth factor-β1; α-smooth muscle actin; hepatocyte growth factor
7,12-Dimethylbenz(a)-anthracene (DMBA)-induced oral buccal mucosa squamous cell carcinoma in Syrian golden hamsters was used to establish precancerous lesions. Agilent rat whole-genome microarray and biological information analysis were used to screen for genes related to key diseases during the transformation of normal buccal mucosa to precancerous lesions in golden hamsters. DMBA acetone solution (0.5%) was used to establish a model of precancerous lesions in oral buccal mucosa in golden hamsters. The results showed that a total of 1331 genes were differentially expressed, including 1278 known, 53 unknown, 747 up-regulated and 584 down-regulated genes. Analysis revealed a total of 14 gene interaction pathways that significantly associated with the 1278 known differentially expressed genes (P<0.05). In conclusion, the occurrence of precancerous lesions in the oral buccal mucosa of golden hamsters was caused by a number of genetic changes that resulted in changes to their respective pathways. Key candidate genes for the formation of precancerous lesions in oral buccal mucosa included Cyp2b13, Orc1L, casp8, CCL5, CXCL12, CCL20, Serping1, P518/Qrfp, F5, TFPI, Vcam1, Fn1, Angpt2, Lcp2, Cxadr, Lyn, Hck, Btk, RGD1564385/fes, Vav1 and IL5ra.
mucosa; precancerous lesions; signaling pathway; gene chip; bio-informatics
The expression of different MUC glycoproteins has helped define cellular lineage in variety of pancreatic neoplasms, and has helped identify distinct carcinogenic pathways such as the intestinal pathway characterized by diffuse/strong MUC2/CDX2 expression in intestinal-type intraductal papillary mucinous neoplasms (IPMNs) and their associated colloid carcinomas (CCs). In this study, the expression profile of MUC6, a pyloric-type mucin, was investigated in both preinvasive and invasive pancreatic neoplasia. Florid papillary (“in-situ”) components of 9 intraductal oncocytic papillary neoplasms (IOPNs), 24 IPMNs, and 7 mucinous cystic neoplasms (MCNs), were analyzed immunohistochemically for MUC6 expression, as were 15 PanINs, 112 usual invasive ductal adenocarcinomas (DAs), and 14 CCs. In PanINs, MUC6 expression was limited to the very early areas of PanIN-1A that typically have pyloric features. Expression was lost in later stages. Similarly, in IOPNs or IPMNs or MCNs, MUC6 expression was detectable in the cystic or flat areas that have pyloric-like histology. However, in the more advanced (papillary) components of these neoplasms, MUC6 expression was mostly limited to the “cuboidal-cell” but was not seen in the “columnar-cell” phenotype: there was diffuse or strong expression in 8/9 IOPN and, relatively weaker but consistent expression in all 6/6 pancreatobiliary-type IPMNs; whereas virtually no expression in villous or intestinal-type IPMNs. The 7/8 gastric or foveolar-type IPMNs were also negative; in the single case with positivity, the labeling was limited to high-grade dysplastic areas. Interestingly, the papillae in MCNs were also mostly negative. Among invasive carcinomas, 39/112 DAs and only 1/14CC expressed MUC6. In DA, the expression did not correlate with survival (P=0.94), or any of the markers of aggressiveness: more than 2-cm tumor size (P=0.76), positive surgical margins (P=0.27), lymph node metastasis (P=0.82), or high grade (P=0.08). In conclusion: (1) The expression of MUC6 in oncocytic and pancreatobiliary-type neoplasms but not in villous or intestinal-type neoplasms supports the presence of a pyloropancreatic pathway distinct from the MUC2/CDX2 expressing intestinal pathway in intraductal papillary neoplasia. (2) MUC6 expression is present in the earliest (nonpapillary) form of any type of preinvasive neoplasia regardless of whether it is PanIN or IOPN or IPMN or MCN suggesting that these entities may share some characteristics early on, but evolve along divergent pathways as they progress.
MUC6; pancreas; intraductal; intraductal papillary mucinous neoplasm; mucinous cystic neoplasm; papilla; pyloric; intestinal
Polo-like kinase 1 (PLK1) has important functions in maintaining genome stability via its role in mitosis. Because PLK1 is up-regulated in many invasive carcinomas, we asked whether it may also play a role in acquisition of invasiveness, a crucial step in transition to malignancy. In a model of metaplastic basal-like breast carcinoma progression, we found that PLK1 expression is necessary but not sufficient to induce invasiveness through laminin-rich extracellular matrix. PLK1 mediates invasion via vimentin and β1 integrin, both of which are necessary. We observed that PLK1 phosphorylates vimentin on Ser82, which in turn regulates cell surface levels of β1 integrin. We found PLK1 to be also highly expressed in preinvasive in situ carcinomas of the breast. These results support a role for the involvement of PLK1 in the invasion process and point to this pathway as a potential therapeutic target for preinvasive and invasive breast carcinoma treatment.
The highest rates of cervical cancer are found in developing countries. Frontline monitoring has reduced these rates in developed countries and present day screening programs primarily identify precancerous lesions termed cervical intraepithelial neoplasias (CIN). CIN lesions described as mild dysplasia (CIN I) are likely to spontaneously regress while CIN III lesions (severe dysplasia) are likely to progress if untreated. Thoughtful consideration of gene expression changes paralleling the progressive pre invasive neoplastic development will yield insight into the key casual events involved in cervical cancer development.
In this study, we have identified gene expression changes across 16 cervical cases (CIN I, CIN II, CIN III and normal cervical epithelium) using the unbiased long serial analysis of gene expression (L-SAGE) method. The 16 L-SAGE libraries were sequenced to the level of 2,481,387 tags, creating the largest SAGE data collection for cervical tissue worldwide. We have identified 222 genes differentially expressed between normal cervical tissue and CIN III. Many of these genes influence biological functions characteristic of cancer, such as cell death, cell growth/proliferation and cellular movement. Evaluation of these genes through network interactions identified multiple candidates that influence regulation of cellular transcription through chromatin remodelling (SMARCC1, NCOR1, MRFAP1 and MORF4L2). Further, these expression events are focused at the critical junction in disease development of moderate dysplasia (CIN II) indicating a role for chromatin remodelling as part of cervical cancer development.
We have created a valuable publically available resource for the study of gene expression in precancerous cervical lesions. Our results indicate deregulation of the chromatin remodelling complex components and its influencing factors occur in the development of CIN lesions. The increase in SWI/SNF stabilizing molecule SMARCC1 and other novel genes has not been previously illustrated as events in the early stages of dysplasia development and thus not only provides novel candidate markers for screening but a biological function for targeting treatment.
Non-small cell lung cancer (NSCLC) is the most common cause of cancer deaths worldwide. The majority of patents presenting with NSCLC have advanced disease, which precludes curative treatment. Early detection and treatment might result in the identification of more patients with early central lung cancer and improve survival. In addition, the study of early lung cancer improves understanding of lung carcinogenesis and might also reveal new treatment targets for advanced lung cancer. Bronchoscopic investigation of the central airways can reveal both early central lung cancer in situ (stage 0) and other preinvasive lesions such as dysplasia. In the current review we discuss the detection of early squamous lung cancer, the natural history of preinvasive lesions and whether biomarkers can be used to predict progression to cancer. Finally we will review the staging and management of preinvasive lung cancer lesions and the different therapeutic modalities that are available.
biomarkers; diagnosis; non-small cell lung cancer; treatment
The aim of the study was to determine whether use of fluorescence bronchoscopy improves the detection of preinvasive neoplastic bronchial lesions. The data of all patients who underwent fluorescence bronchoscopy and in whom bronchial biopsies were taken, were analyzed. Most patients were at risk for preinvasive lesions. A total of 174 bronchoscopies were performed in 95 patients. Of the 681 representative biopsies, 31 were found to be moderate dysplastic, 39 were found to be severe dysplastic and 9 exhibited carcinoma in situ. These 79 preinvasive lesions were found in 34 patients. The respective results of fluorescence bronchoscopy in addition to conventional bronchoscopy and of conventional bronchoscopy alone for detection of preinvasive lesions were: sensitivity 85% (67/79) and 59% (47/79); specificity 60% (351/581) and 85% (493/581); positive predictive values of 23% (67/297) and 35% (47/135); negative predictive values of 97% (351/363) and 94% (493/525). A separate analysis of only the first bronchoscopy of each patient showed similar results. Results of fluoresence bronchoscopy were better in the second part of the patient group. We conclude that after a learning period fluorescence bronchoscopy can increase the yield of finding preinvasive neoplastic lesions when used in addition to conventional bronchoscopy.
Advances in molecular biology have increased our knowledge of the biology of preneoplastic lesions in the human lung. The recently published WHO lung tumour classification defines three separate lesions that are regarded as preinvasive neoplasia. These are (1) squamous dysplasia and carcinoma in situ (SD/CIS), (2) atypical adenomatous hyperplasia (AAH), and (3) diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH). SD/CIS is graded in four stages (mild, moderate, severe, and CIS), based upon the distribution of atypical cells and mitotic figures. Most airways showing SD/CIS demonstrate a range of grades; many epithelia are hard to assess and the reproducibility of this complex system remains to be established. Detailed criteria are, however, welcome and provide an objective framework on which to compare various molecular changes. Alterations in gene expression and chromosome structure known to be associated with malignant transformation can be demonstrated in CIS, less so in dysplasias, but also in morphologically normal epithelium. The changes might be sequential, and their frequency and number increase with atypia. Less is known of the "risk of progression" of SD/CIS to invasive "central" bronchial carcinoma. It may take between one and 10 years for invasion to occur, yet the lesion(s) may be reversible if carcinogen exposure ceases.
AAH may be an important precursor lesion for peripheral "parenchymal" adenocarcinoma of the lung: the "adenoma" in an adenoma–carcinoma sequence. There is good morphological evidence that AAH may progress from low to high grade to bronchioloalveolar carcinoma (BAC; a non-invasive lesion by definition). Invasion then develops within BAC and peripheral lung adenocarcinoma evolves. The molecular events associated with this progression are not well understood and studies are hampered by a lack of clear criteria to distinguish high grade AAH from BAC. Nonetheless, as with SD/CIS, the patterns of expression of tumour associated genes are consistent with neoplastic progression. We have little idea of the incidence of AAH in the normal or "smoking" populations. It is found more frequently in cancer bearing lungs, especially in those with adenocarcinoma, and is more common in women. No data are available on the risk of progression of AAH. DIPNECH is an exceptionally rare lesion associated with the development of multiple carcinoid tumours. Almost nothing is known of its biology.
Knowledge of these lesions will be crucial in the design and understanding of lung cancer screening programmes, where it is likely that the morphological and, more importantly perhaps, the molecular characteristics of these lesions will provide useful targets for detection and possibly even treatment.
Key Words: lung cancer • preneoplasia • carcinogenesis
This study aimed to screen the pathogenic genes and pathways that relate to the transformation of hamster buccal mucosa from precancerous lesions to squamous cell carcinoma by whole genome microarray and bioinformatics analysis. A DMBA (7,12-dimethylbenz[a]anthracene)-induced hamster model of a precancerous lesion and squamous cell carcinoma was established. The differentially expressed genes were detected using an Agilent whole rat genome microarray, which contains 41,000 genes/ESTs. Gene ontology (GO) functional classification and pathway analyses were performed, and a subset of differentially expressed genes were validated using RT-PCR. The results showed that during the transformation of hamster buccal mucosa from the precancerous lesion to squamous cell carcinoma, a total of 1,981 genes were differentially expressed, of which 1,037 were up-regulated and 944 were down-regulated. GO analysis revealed that the differentially expressed genes are mainly involved in 14 functional groups including those of metabolism and cell structure. Additionally, 9 significantly altered pathways were identified. Among the 1,861 known differentially expressed genes, 14 genes including Casp3, CCL5 and CXCL12 were enriched in the 9 altered pathways. The up-regulation of SPARC and down-regulation of Casp3 were confirmed by RT-PCR. In conclusion, a total of 1,981 differentially expressed genes and 9 significantly altered pathways were identified in the transformation of hamster buccal mucosa from precancerous lesions to squamous cell carcinoma. A total of 14 pathway-enriched genes including Casp3, CCL5 and CXCL12 may play critical roles in the alteration of cellular pathways leading to the transformation of buccal mucosa from precancerous lesions to squamous cell carcinoma. Future studies focusing on these genes and pathways are required in order to gain a better understanding and provide effective prevention and treatment of oral squamous cell carcinoma.
oral; precancerous lesion; squamous cell; carcinoma; gene; pathway
Lung cancer is the most common cause of cancer-related deaths. Tobacco smoke exposure is the strongest aetiological factor associated with lung cancer. In this study, using serial analysis of gene expression (SAGE), we comprehensively examined the effect of active smoking by comparing the transcriptomes of clinical specimens obtained from current, former and never smokers, and identified genes showing both reversible and irreversible expression changes upon smoking cessation.
Twenty-four SAGE profiles of the bronchial epithelium of eight current, twelve former and four never smokers were generated and analyzed. In total, 3,111,471 SAGE tags representing over 110 thousand potentially unique transcripts were generated, comprising the largest human SAGE study to date. We identified 1,733 constitutively expressed genes in current, former and never smoker transcriptomes. We have also identified both reversible and irreversible gene expression changes upon cessation of smoking; reversible changes were frequently associated with either xenobiotic metabolism, nucleotide metabolism or mucus secretion. Increased expression of TFF3, CABYR, and ENTPD8 were found to be reversible upon smoking cessation. Expression of GSK3B, which regulates COX2 expression, was irreversibly decreased. MUC5AC expression was only partially reversed. Validation of select genes was performed using quantitative RT-PCR on a secondary cohort of nine current smokers, seven former smokers and six never smokers.
Expression levels of some of the genes related to tobacco smoking return to levels similar to never smokers upon cessation of smoking, while expression of others appears to be permanently altered despite prolonged smoking cessation. These irreversible changes may account for the persistent lung cancer risk despite smoking cessation.
Twenty-three cases of ductal carcinoma in situ (DCIS), ten of which had an associated invasive component, were studied for loss of heterozygosity (LOH) of microsatellite markers on chromosome 9p and the results compared with a panel of 20 invasive breast carcinomas. In addition to the gene encoding p16, chromosome 9p is also thought to contain other putative tumour-suppressor genes. If the three panels of breast tumours showed LOH of markers in this region this would suggest that such putative genes were important in breast carcinogenesis. By studying both preinvasive and invasive breast tumours, it should also be possible to gain further information about the relationship between lesions of a different stage and to determine whether DCIS is indeed a precursor of invasive ductal carcinoma. Levels of LOH were low in the invasive-only set of tumours. Surprisingly, considerably higher levels of loss were observed in the tumours with an in situ component. Also, much heterogeneity was observed between different DCIS ducts or invasive tumour and DCIS from the same case.
FHIT and WWOX are tumor suppressor genes that span the common fragile sites FRA3B and FRA16D, respectively. To analyze possible synergisms among these genes in cervical cancer progression, we considered 159 cervical intraepithelial neoplasias, and 58 invasive squamous cell carcinomas of the uterine cervix. FHIT and WWOX proteins were examined by immunohistochemistry and their expression was inversely correlated with precancerous versus invasive lesions. Statistics among biological markers indicated a strong association between FHIT and WWOX. Protein expression of these two genes was also absent or reduced in cancer cell lines. Thus, WWOX may be considered as a novel important genetic marker in cervical cancer and the association between the altered expression of FHIT and WWOX may be a critical event in the progression of this neoplasia.
Cervical neoplasia; immunohistochemistry; tumor suppressors; fragile sites
Squamous epithelial dysplasia is often observed multifocally in the cancerous oesophagus and is presumably considered to be a pre-cancerous lesion. A mutation of the p53 tumour suppressor gene is commonly identified in oesophageal cancer and dysplasia. p53 mutations can be anticipated immunohistochemically. In order to confirm the biological and clinical significance of p53 expressions in oesophageal field carcinogenesis, immunostaining for p53 in cancerous and multifocal precancerous lesions from resected human oesophagus was systematically investigated, while paying special attention to the contiguity of these lesions. Lesions expressing p53 were detected in 46.5% (20 of 43 lesions) of the invasive carcinoma, and in 51.0% (46 of 90 lesions) of the carcinoma in situ, and in 51.4% (92 of 179 lesions) of the dysplasia. Next, the p53 expression in dysplasia was compared with that in carcinoma for the same case. 37 of 39 (94.8%) dysplasias contiguous to p53-positive carcinomas also expressed p53 (P < 0.0001). On the other hand, the isolated dysplasias without contiguity to p53-positive carcinomas, only expressed p53 protein in 44.0% (11 of 25 lesions). No significant correlations were found between the p53 staining and either the clinicopathological features or prognosis. Discordant p53 alterations, such as those seen in cancerous and isolated precancerous lesions, may thus demonstrate further evidence for a multicentric or field carcinogenesis of the human oesophagus. © 2000 Cancer Research Campaign
dysplasia; oesophagus; p53; immunohistochemistry; field carcinogenesis; prognosis
A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from preinvasive to invasive phenotype as it may occur “spontaneously” in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted basement membrane cultures. These cells remained noninvasive; however, unlike their nonmalignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher-grade tumors. To find functionally significant changes in transition from preinvasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between preinvasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP9, MMP13, MMP15, and MMP17 was up-regulated in the invasive cells. Using small interfering RNA–based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which preinvasive breast cells could acquire invasiveness in a metaplastic context.
Progression through G1 phase of the eukaryotic cell cycle is tightly controlled by cyclin-dependent kinases (CDK). These proteins form part of a regulatory pathway including the cyclin-dependent kinase inhibitor (CKI) p16, D-type cyclins and the product of the retinoblastoma gene pRb. Aberration of any one of these components may lead to uncontrolled proliferation contributing to neoplasia. Three of these proteins, cyclin D1, pRb and p16, were analysed by immunohistochemistry on archival paraffin sections to determine whether expression patterns were different in preinvasive ductal carcinoma in situ (DCIS) and invasive breast tumours relative to normal. Genetic analysis of the gene encoding cyclin D1 (CCND1) was also carried out, using an intragenic restriction fragment-length polymorphism (RFLP) to assess possible allelic imbalance. A majority of the tumours studied (approximately 90%) showed abnormalities in expression of at least one of these proteins. Overexpression of cyclin D1 was found in approximately 49% cases, reduced expression of p16 in approximately 46% and reduced expression of pRb in approximately 37%. Allelic imbalance of cyclin D1 was found in approximately 57% cases.