Pre-invasive Precursor Research Articles

Abstract 5457: High levels of fatty acid synthase expression in esophageal cancers represent a potential target for therapy

Abstract Fatty acid synthase (FAS) is overexpressed in many human cancers and is considered to be a promising target for therapy. To investigate the expression of this candidate target in esophageal cancer, we evaluated expression of FAS protein in - cases of esophageal squamous cancer, - cases of esophageal adenocarcinoma, and - cases of Barrett's esophagus with dysplasia - a lesion thought to represent a pre-invasive precursor to esophageal cancer. Using immunohistochemistry, we found significantly higher levels of FAS expression in -% of the squamous cancers and -% of the adenocarcinomas compared to normal esophageal epithelium. To evaluate the potential for inhibiting this enzyme as a treatment of esophageal cancer, we treated mice bearing xenografts of the Colo680N (squamous carcinoma) and Bic1 (adenocarcinoma) cell lines using C93, a rationally designed molecule that inhibits FAS activity. In these experiments, C93 significantly inhibited the growth of orthotopic xenograft tumors from without causing anorexia and weight loss in the treated animals. We conclude that, similar to several other common types of human cancer, FAS is expressed at very high levels in esophageal cancer, and growth of these cancers can be inhibited by pharmacological agents that target this enzyme. Moreover, this high expression of FAS is also seen in pre-invasive lesions of the esophagus, leading us to propose considering of FAS-inhibitors for purposes of esophageal cancer chemoprevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5457.

Epithelioid melanoma of the rectum: reappraisal of a deceptive microscopic simulator

We report 2 cases of rectal melanoma. The patients were a 48-year-old man and an 88-year-old woman. In both cases, the tumor presented as polypoid and necrotic masses, making initial diagnostic assessment difficult because of the regressive tissue changes. Microscopically, tumors were mostly composed of epithelioid, highly atypical cells mimicking poorly differentiated carcinoma or large cell lymphoma. Tumor cells were negative for keratin, leukocyte common antigen, and epithelial membrane antigen and positive for S100 protein and several melanoma markers, although in endoscopic biopsy specimens, the positive reaction was focal due to the limited viable tissue. One patient was treated with abdominoperineal resection. Pathologic examination of the surgical specimen revealed a polypoid mass infiltrating the muscle coat of the rectum. Local lymph nodes contained metastatic deposits. Microscopic examination of the rectal mucosa adjacent to the infiltrating neoplasm revealed colonization of the intestinal crypts by atypical melanocytes. This patient is alive 1 year after surgery with no evidence of recurrent disease. The other case was not deemed amenable to radical surgery because of the patient's advanced age and evidence of diffuse metastatic disease. She was alive 6 months after the initial diagnosis but was lost to follow-up. Data indicate that the diagnosis of melanoma, although feasible in large resection specimens, may be problematic in endoscopic biopsy, especially when only small tissue fragments are available and tumor regression hampers the accuracy of immunohistochemical stains. Documentation of intracryptic atypical melanocytes in nonneoplastic mucosa conceptually corroborates claims that rectal melanoma is related to preinvasive precursors as in cutaneous melanoma.

Differential developmental expression of transcription factors GATA-4 and GATA-6, their cofactor FOG-2 and downstream target genes in testicular carcinoma in situ and germ cell tumors

Testicular germ cell cancer is the most common malignancy among young males. The pre-invasive precursor, carcinoma in situ testis (CIS), presumably originates from arrested and transformed fetal gonocytes. Given that GATA transcription factors have essential roles in embryonic and testicular development, we explored the expression of GATA-4, GATA-6, cofactor friend of GATA (FOG)-2, and downstream target genes during human testis development and addressed the question whether changes in this pathway may contribute to germ cell neoplasms. Fetal testis, testicular CIS, and overt tumor samples were analyzed by immunohistochemistry for GATA-4, GATA-6, FOG-2, steroidogenic factor 1 (NR5A1/SF1), anti-Müllerian hormone/Müllerian-inhibiting substance (AMH), and inhibin-alpha (INHalpha). GATA-4 was not expressed in normal germ cells, except for a subset of gonocytes at the 15th gestational week. The CIS cells expressed GATA-4 and GATA-6 heterogeneously, whereas most of the CIS cells expressed GATA-4 cofactor FOG-2. GATA target gene SF-1 was expressed heterogeneously in CIS cells, whereas INHalpha and AMH were mostly negative. Seminomas and yolk sac tumors were positive for GATA-4 and GATA-6, but mostly negative for FOG-2 and the GATA target genes. In contrast, pluripotent embryonal carcinomas and choriocarcinomas were GATA-4 and GATA-6 negative. Differential expression of the GATA-4 target genes suggested cell-specific functions of GATA-4 in the germ and somatic cells. The GATA-4 expression in early fetal gonocytes, CIS, and seminoma cells but the absence in more mature germ cells is consistent with the early fetal origin of CIS cells and suggests that GATA-4 is involved in early germ cell differentiation.

Identification of ERβ1 and ERβ2 in human seminoma, in embryonal carcinoma and in their adjacent intratubular germ cell neoplasia

BackgroundEstrogens exert a role on germ cell physiology of normal human testis through the mediation of the estrogen receptor (ER) beta subtypes. Epidemiological studies evidenced an increased incidence of testicular germ cell cancer after elevated pre-natal estrogen exposure but the expression of estrogen receptors in these testicular neoplasms has not been well elucidated.MethodsImmunohistochemistry and Western blot analysis were used to investigate the expression of three distinct ER isoforms, ERalpha, ERbeta1, and ERbeta2 in paraffin-embedded tissues from seminomas and embryonal carcinomas, which are the most common testicular germ cell tumours.ResultsNeoplastic cells of all specimens revealed a positive ERbeta1 and ERbeta2 immunoreactivity, while the ERalpha signal was undetectable. A similar pattern of estrogen receptor immunostaining was also observed in the malignant germ cells of intratubular germ cell neoplasia, adjacent to testicular cancers. Western blot analysis of tumour extracts revealed two immunoreactive bands, a 59 kDa band for ERbeta1 and a 53 kDa band for ERbeta2.ConclusionA variable ERbeta expression was previously reported in testicular germ cell tumours and, particularly, an ERbeta down-regulation was evidenced in seminoma and embryonal carcinoma. Conversely, the current study has clearly identified ERbeta1 and ERbeta2 in the neoplastic cells of seminoma and embryonal carcinoma, as well as in the malignant cells of their common pre-invasive precursor, intratubular germ cell neoplasia. Therefore, our findings suggest that ERbeta1, together with a possible ERbeta2 contribute, can mediate estrogen action in both early and late neoplastic testicular germ cells, not confirming the previously hypothesized antiproliferative effect of ERbeta on male gonadal cells.

Novel cell culture model for prevention of carcinogenic risk in familial adenomatous polyposis syndrome

Clinical familial adenomatous polyposis (FAP) syndrome represents a high risk pre-invasive precursor for colon cancer, and is characterized by germ line mutation in the adenomatous polyposis coli (APC) tumor suppressor gene. Cellular models with relevant genetic and biological characteristics should provide important mechanistic leads for predisposition and preventive intervention. Cloned colon epithelial cell line from the Apc850 Min/+ mouse represented a model for FAP. Cell cycle progression, cellular apoptosis and anchorage-independent growth represented the biomarkers for carcinogenic risk. The Apc mutant 850Min COL-Cl1 cells exhibited decreased G0/G1:S+G2/M ratio, increased S+G2/M:subG0 ratio, and increased anchorage-independent colony formation, indicating loss of homeostatic growth control and gain of anchorage-independent growth. Growth of these cells in serum-depleted medium was promoted by mitogenic insulin and epidermal growth factor, and inhibited by anti-mitogenic transforming growth factor-beta1 and dexamethasone. Treatment with low dose combinations of synthetic enzyme inhibitor difluoro methylornithine (DFMO), synthetic non-steroidal anti-inflammatory drug sulindac (SUL), and naturally occurring epigallocatechin gallate (EGCG), and eicosapen-taenoic acid (EPA) produced cytostatic growth arrest and inhibited anchorage-independent colony formation. These data identify a novel cell culture model and validate a mechanism-based approach to prioritize combinations of effective chemopreventive compounds for prevention/therapy of colon cancer.

From Gonocytes to Testicular Cancer

Testicular germ-cell tumors occur primarily in young individuals, and the tumors in this age group (seminomas or nonseminomas) are derived from a preinvasive precursor cell called carcinoma in situ (CIS) or intratubular germ-cell neoplasia. These tumors have been a growing problem, especially in highly developed industrialized countries. A hypothesis was put forward that CIS originates from arrested fetal germ cells, thus testicular cancer is a developmental disease of germ-cell differentiation. This notion was supported by comparative studies of the gene expression at the protein and RNA level, which demonstrated a close similarity of CIS to primordial germ cells and gonocytes with many features of embryonic stem cells. The arrest of germ-cell differentiation is thus the key first event, which may be followed by malignant transformation and overt germ-cell cancer in young adult age, usually after puberty. In most cases the arrest/delay of germ-cell differentiation is caused by testicular dysgenesis, a multifactorial and complex syndrome that has a broad spectrum of phenotypes ranging from moderate impairment of spermatogenesis to severe disorders of sexual development and differentiation. The most severe cases are a result of inherited genetic aberrations, but the etiology of the common sporadic testicular cancer must involve environmental factors, including maternal lifestyle and possibly an early exposure to endocrine disruptors. The effects of environmental factors are likely modulated by genomic variation (polymorphisms), thus explaining the individual susceptibility and population-level differences in the incidence of testicular cancer.

Preinvasive intraductal neoplasia in salivary adenocarcinoma, not otherwise specified

Preinvasive intraductal neoplasia of the salivary glands has only been identified in the rare salivary-duct carcinoma, whereas, it is an established feature of carcinomas of other glands. A fortuitous observation of what appeared to be intraductal tumor in a salivary adenocarcinoma, not otherwise specified, led to the present investigation to determine whether intraductal neoplasia is a significant feature of this carcinoma. Intraductal tumor confined by normal CK14-positive, actin-negative ductal basal cells was identified in 15 of 22 cases (68%). The degree of cellular atypia and the pattern of growth of intraductal tumor was similar to that of the invasive tumor. Cases with intraductal tumor devoid of invasive tumor were not found. Intraductal tumor is identified as the pre-invasive precursor of adenocarcinoma, not otherwise specified, and apparently develops in excretory ducts. The findings support the possibility that different salivary tumors arise from different types of parenchymal cell. Possibly intraductal neoplasia is a universal feature of many types of salivary tumor, but has been overlooked because of the need to use immunohistology to demonstrate it and because it may no longer be present as such when the tumor presents as a clinical lesion.

Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects

Carcinoma in situ testis (CIS), also known as intratubular germ cell neoplasia (ITGCN), is a pre-invasive precursor of testicular germ cell tumours, the commonest cancer type of male adolescents and young adults. In this review, evidence supporting the hypothesis of developmental origin of testicular germ cell cancer is summarized, and the current concepts regarding aetiology and pathogenesis of this disease are critically discussed. Comparative studies of cell surface proteins (e.g. PLAP and KIT), some of the germ cell-specific markers (e.g. MAGEA4, VASA, TSPY and NY-ESO-1), supported by studies of regulatory elements of the cell cycle (e.g. p53, CHK2 and p19-INK4d) demonstrated a close similarity of CIS to primordial germ cells and gonocytes, consistent with the pre-meiotic origin of CIS. Recent gene expression profiling studies showed that CIS cells closely resemble embryonic stem cells (ESCs). The abundance of factors associated with pluripotency (NANOG and OCT-3/4) and undifferentiated state (AP-2gamma) may explain the remarkable pluripotency of germ cell neoplasms, which are capable of differentiating to various somatic tissue components of teratomas. Impaired gonadal development resulting in the arrest of gonocyte differentiation and retention of its embryonic features, associated with an increasing genomic instability, is the most probable model for the pathogenesis of CIS. Genomic amplification of certain chromosomal regions, e.g. 12p, may facilitate survival of CIS and further invasive progression. Genetic studies, have so far not identified gene polymorphisms predisposing to the most common non-familial testicular cancer, but this research has only recently begun. Association of CIS with other disorders, such as congenital genital malformations and some forms of impaired spermatogenesis, all rising in incidence in a synchronous manner, led to the hypothesis that CIS might be a manifestation of testicular dysgenesis syndrome (TDS). The aetiology of TDS including testicular cancer remains to be elucidated, but epidemiological trends suggest a primary role for environmental factors, probably combined with genetic susceptibility.

Pancreatic intraepithelial neoplasia.

Great efforts have been devoted to detecting preinvasive precursors to ductal carcinoma of the pancreas in the hope of improving the currently bleak prognosis of invasive pancreatic cancer. Intensive investigations of the pancreas have led to the recognition of intraductal papillary mucinous neoplasms (IPMNs) and the detection of preinvasive precursors to conventional ductal carcinoma. The pancreatic intraepithelial neoplasia (PanIN) nomenclature for precursor lesions of ductal carcinoma was developed at the "Pancreatic Cancer Think Tank" held in the United States in 1999. However, some reports of precursor lesions have suggested that these definitions do not encompass the full spectrum of precursors of ductal carcinoma, and these issues were the subject of the "Forum on Carcinoma In Situ of the Pancreas" held in Japan in 2002. After this forum, it became clear that the existing definitions of PanINs needed to be revised and expanded. Both participants of the Pancreatic Cancer Think Tank and the Forum gathered together at a meeting on precursor lesions of pancreatic cancer in 2003, and an international consensus on the diagnostic criteria for PanINs and IPMNs was created. We describe herein the current understanding of precursor lesions of pancreatic cancer.

Positive correlation between the frequency of micronucleated cells and dysplasia in Papanicolaou smears.

Most cancers arise in epithelial tissues and these tissues are typically the targets of the carcinogens responsible for the tumors. Exfoliated epithelial cells have traditionally been used for cancer screening by cytopathologists and these cells also can be used for biomonitoring of genotoxic effects in humans. Cervical cancer results from the progression of preinvasive precursor lesions, called low-grade squamous intraepithelial lesions (LGSILs), to high-grade squamous intraepithelial lesions (HGSILs). The gradient from low to high-grade lesions is characterized by increasing nuclear atypia and the failure of cellular differentiation in progressively more superficial levels of the epithelium. These phenotypic changes are hypothesized to be accompanied by increased genetic instability that can be documented using the micronucleus (MN) assay in exfoliated cervical cells. A retrospective study was performed to investigate the frequency of micronucleated cells in cervical smears from women at high risk for developing cervical cancer. Papanicolaou (Pap) smears from 275 women previously studied at a cancer clinic were coded and analyzed for the frequency of micronucleated cells. LGSIL, HGSIL, and invasive carcinoma smears had significantly higher frequencies than normal and ASCUS (abnormal squamous cells of undetermined origin) smears. HGSIL or severely dysplastic smears had the highest frequency of micronucleated cells (although not significantly higher than LGSIL smears), an observation that that could be useful in confirming these types of lesions. The results indicate that the MN frequency in exfoliated cervical cells may be an additional criterion for establishing cervical cancer risk.

Soy isoflavone genistein modulates cell cycle progression and induces apoptosis in HER-2/neu oncogene expressing human breast epithelial cells

In the multistep progressive pathogenesis of human breast cancer, comedo ductal carcinoma in situ (DCIS) represents a preinvasive precursor lesion for therapy resistant invasive cancer. Human tissue derived cell culture models exhibiting molecular similarities to clinical DCIS facilitate an important preclinical mechanistic approach for evaluation of preventive efficacy of natural and synthetic chemopreventive compounds. Natural phytochemicals present in fresh fruits, vegetables and grain products are likely to offer protection against cancer. The clinical efficacy of these natural phytochemicals, however, depends on extrapolation, and is therefore equivocal. The present study determined whether the natural soy isoflavone genistein (GEN) inhibited aberrant proliferation in 184-B5/HER cells (a model for human comedo DCIS) and identified possible mechanisms responsible for its efficacy. Human reduction mammoplasty derived HER-2/neu oncogene expressing preneoplastic 184-B5/HER cells represented the experimental system. Flow cytometry and cellular epifluorescence based assays were utilized to quantitate the alterations in cell cycle progression, cellular apoptosis, and in the status of cell cycle regulatory and apoptosis-associated gene product expression. The 184-B5/HER cells exhibited specific immunofluorescence to p185HER, p53, EGFR, but not to ERalpha, thus resembling comedo DCIS. Treatment of 184-B5/HER cells with GEN resulted in a dose-dependent decrease in the viable cell population, increase in the G0/G1:S + G2/M ratio and enhancement of sub G0/G1 (apoptotic population). Exposure to the maximum cytostatic 10 microM dose of GEN down-regulated HER-2/neu mediated signal transduction as evidenced by a 73.9% decrease (p=0.001) in p185HER specific, and a 89.8% decrease (p=0.001) in phosphotyrosine specific immunofluorescence. The increase in G0/G1:S + G2/M ratio in response to the treatment with 10 microM GEN was associated with a 85.5% decrease (p=0.001) in immunoreactivity to PCNA and a 128.6% increase (p=0.004) in immunoreactivity to the cyclin dependent kinase inhibitor p16INK4. The induction of apoptosis by GEN was associated with a 52.8% decrease (p=0.001) in the immunoreactivity to antiapoptotic Bcl-2 and with a 195.9% (p=0.001) increase in the immunoreactivity to proapoptotic Bax. Thus, preventive efficacy of GEN in HER-2/neu+/ER- 184-B5/HER cells may be due to its ability to down-regulate HER-2/neu mediated signal transduction, increase the expression of the cyclin dependent kinase inhibitor p16INK4, and induce Bcl-2 dependent apoptosis. These data provide evidence that GEN may be a potential chemopreventive lead compound for human comedo DCIS. The 184-B5/HER cells, may therefore, provide a high throughput mechanistic bioassay to identify new chemopreventive agents for human breast cancer.

Positive immunohistochemical staining of p53 and cyclin D in advanced mouse skin tumors, but not in precancerous lesions produced by benzo[a]pyrene.

Squamous cell carcinomas (SCCs) of the mouse skin, as well as several types of preinvasive carcinoma precursor lesions, were produced by complete carcinogenesis protocols with benzo[a]pyrene (B[a]P). Groups of mice were studied histologically at several time points. Tumors and precursor lesions were systematically counted on microscope slides. The main feature of tumor development using this ubiquitous human carcinogen was the sequential appearance of in situ flat lesions with progressive degrees of dysplasia. These changes, preceding the development of SCCs, were observed 20 weeks after beginning the carcinogen treatments. At this time point, in situ lesions outnumbered SCC approximately 10:1 at the higher total carcinogen dose examined. Ten weeks later, this ratio was approximately 1:1. With the lower total carcinogen dose protocol, progression was delayed since at 27 weeks preinvasive lesions outnumbered SCCs approximately 8:1. In addition to the in situ lesions, papillomas and keratoacanthomas were noted with the high B[a]P dose protocol, but tended to disappear at the end of the experiment, also indicating their probable role as SCC precursors. A study of histochemical markers showed that gamma-glutamyltranspeptidase (GGT) and keratin 13, although good markers of malignant changes in early papillomas produced by two-stage carcinogenesis protocols, were mainly negative in dysplastic lesions produced by complete carcinogenesis with B[a]P. Immunohistochemical detection of p53 showed that 50% of SCCs were positively stained, whereas only 3% of in situ lesions were p53 immunoreactive. Similarly, 62% of SCCs were immunohistochemically positive for cyclin D, but no precursor lesions were positive. Molecular analysis of the tumors showed the absence of H-ras mutations. No amplification of the cyclin-D-1 gene was detected in eight SCCs examined. Collectively, these findings indicate that preinvasive in situ lesions are frequent during early stages of carcinogenesis when B[a]P is used in a complete carcinogenesis protocol. Although the absence of p53 immunoreactivity in this mouse model differs from the observed changes in human premalignant squamous lesions, the sequence of morphological changes and the final incidence of p53 and cyclin D staining abnormalities are very similar to the well-known alterations that take place during human squamous carcinogenesis.

Fortschritte in der Früherkennung des Zervixkarzinoms aus der Sicht des saarländischen Krebsregisters

The efficacy of the nationwide screening programme for cervical cancer in the Federal Republic of Germany, which has been in effect since 1971, has never been checked by means of controlled epidemiological studies. Therefore routinely collected mortality and morbidity data are up to now the only indicators of potential progress in early detection. Mortality statistics of cervical cancer are of restricted value due to lack of specificity regarding the cause of death on death certificates. Data of the population based cancer registry of Saarland are used to investigate trends in terms of age, stage and birth cohort-specific detection rates of cervical cancer and its preinvasive precursors. There was a substantial decrease in incidence rates of invasive cervical cancer, which was most pronounced for advanced tumour stages and young and middle-age groups and which is consistent with comparable results in other countries following the introduction of screening programmes. However, a selection effect of the screening programme suggested by a decrease in survival rates of women with invasive cervical cancer in the 1980ies, must also be taken into account.

Eccrine porocarcinoma: A case report with light microscopy and ultrastructure

A case of eccrine porocarcinoma in a 63-yr-old man is reported. There was a 10 yr history of an abdominal skin lesion which recurred after cautery and metastasized to skin and abdominal lymph nodes. Histologically, the primary tumour was composed of small squamoid cells with ductal lumina. There was intraepidermal nesting spread and an invasive dermal component. Formation of intracytoplasmic lumina in cells with squamoid features, best seen on electron microscopy, pointed towards poral differentiation and was a useful diagnostic feature which persisted in the less well differentiated metastases. The role of hidroacanthoma simplex as a possible preinvasive precursor lesion is discussed. The differential diagnosis from other skin tumours is described. The literature is reviewed. Despite their sometimes innocuous histological appearance, eccrine porocarcinomas are aggressive lesions which require wide local excision of the primary site and consideration of regional lymph node dissection.