Preinvasive Tumors Research Articles

Cell budding from pre-invasive tumors: Intrinsic precursor of invasive breast lesions?

Our previous studies showed that in patients with ductal carcinoma in situ (DCIS) of the breast, the tumor cells that overlie focal myoepithelial cell layer disruptions (FMCLDs) are generally arranged as finger-like projections that bud into the stroma. These budding cells have significantly more genetic instability and invasion-related gene expression, and less estrogen receptor (ER) expression, than their epithelial cell counterparts. This study aimed to assess these cells for potential molecular markers that are uniquely associated with cell adhesion and motility. Seventeen ER-positive DCIS cases were screened by immunostaining for ER, and 7 cases which harbored FMCLD lesions were used to examine the expression of the potential markers. Two cases with both DCIS and invasive lesions were selected for comparing the differences in molecular expression between these lesion types. The results showed that expression levels of talin, E-cadherin and focal adhesion kinase (FAK) in tumor cells overlying FMCLDs were higher than those within the corresponding duct. Integrin β1 staining was detected only in a small number of the tumor cells overlying the FMCLDs. Vinculin staining was weak (18%) or not detected (82%), and no expression was found in the tumor cells within the corresponding duct or in the pure isolated DCIS. By contrast, the expression levels of talin, vinculin and integrin β1 in the invasive tumors were distinctly higher than those in DCIS, and the expression of FAK and E-cadherin was lower. Using electron microscopy, we found that the tight junctions between tumor cells overlying the FMCLDs were reduced compared to the adjacent tumor cells in the lumen. These results indicate that the tumor cells overlying FMCLDs are likely to represent the specific precursors of invasive breast lesions. Our findings may also facilitate the identification of specific targets for further molecular profiling, which will more completely characterize this important cell population.

Ezrin expression in primary breast cancer, metastatic lymph nodes and normal breast tissue

Aim: To investigate the ezrin expression in normal breast tissue, primary breast cancer and lymphnode metastases and study the correlation between ezrin expression in breast cancer tissue, tumor invasion and metastasis. Methods: Immunohistochemistry was used to detect the ezrin expression in normal breast tissue, breast cancer tissue and metastatic lymph nodes using two commercially available tissue arrays BR2082 and BR1005. The tissue array BR1005 consists of 50 samples of primary breast cancer and 50 samples of lymph node metastases while the tissue array BR2082 includes 32 metastatic lymph node samples, 94 invasive breast cancer samples, 26 preinvasive breast tumors, 40 benign breast tumors and 16 normal breast tissue samples. Results: We found a statistically significant difference between the ezrin expression in benign breast tumors and breast cancer samples (chi square test: p<0.0001). There was no significant difference between the expression of ezrin in metastatic lymph nodes and primary breast cancer (p=0.4899). Conclusion: Ezrin expression is obviosly higher in malign than in benign breast tumors correlating with the hypothesis of ezrin playing an important role in the tumor invasion process. We could not show a correlation between the expression of ezrin and the process of metastasis.

Abstract 2727: Predicting lung cancer risk based on a novel proteomic signature of bronchial lesions

Abstract A biomarker signature to identify individuals at-risk for lung cancer would be of great clinical value. We hypothesized that the bronchial epithelium expresses a subset of proteins that once increased confer an elevated risk for lung cancer. We have therefore developed a proteomic approach to assess the risk for lung cancer. From our previous studies, we selected 11 matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) features to distinguish normal bronchial epithelium and low grade preinvasive lesions from high grade preinvasive lesions and invasive lung tumors. We tested this signature in an independent validation set consisting of bronchial specimens from 60 patients. The prediction accuracy was 78% with 73% sensitivity and 81% specificity. There was a 19-fold increased risk of having lung cancer for individuals with a signature intensity above the third quartile (95% CI, 5.65-64.81). We identified 10 of the 11 features corresponding to 7 proteins, thymosin ß4, ubiquitin, acyl-coA binding protein, cystatin A, S100A11, cytochrome c and macrophage migration inhibitory factor. The identity of these proteins was validated by immunohistochemistry, Western blotting and MALDI imaging mass spectrometry (IMS). MALDI IMS localized the candidate biomarker proteins specifically to high grade preinvasive lesions and invasive tumors. This novel proteomic signature may facilitate the selection and monitoring of high risk individuals for lung cancer early detection and chemoprevention strategies. 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 2727.

Abstract 2250: Aberrant leukocyte infiltration as a trigger for tumor metastasis

Abstract Our previous studies have led to a novel hypothesis that aberrant leukocyte infiltration is a trigger for tumor invasion. Our hypothesis and supporting data have been published in multiple peer-reviewed journals. Our current studies attempted to further expand our previous observations, and to test a novel hypothesis that aberrant leukocyte infiltration may also represent a trigger for tumor metastasis. Tissue sections from human breast, prostate, cervical, and lung tumors were analyzed with multidisciplinary approaches with a panel of tumor metastasis-related biomarkers. Our study revealed that pre-invasive tumors surrounded by or immediately adjacent to leukocyte aggregates displayed a number of unique features: [1] significantly reduced expression of cell surface adhesion molecules, [2] significantly elevated blood vessels and lymphatic ducts at the intersection between the tumor core and leukocyte aggregates, [3] the presence of isolated individual tumor cells or cell clusters within the leukocyte aggregates, [4] physical attachment or fusion of tumor cell membrane and condensed chromosomes of mitotic tumor cells with leukocytes, and [5] the presence of co-joined tumor cells and leukocytes within vascular structures or lymphatic ducts. Together, our findings suggest that leukocyte infiltration may trigger tumor metastasis through three correlated mechanisms: (1) to physically disrupt the inter-cellular junctions and cell surface adhesion molecules, [2] to interfere with the mitotic process or physically drag tumor cells to different sites during their migration, and [3] to stimulate growth of vascular structures that facilitate tumor metastasis. (Supported in part by grants DAMD17-01-1-0129, DAMD17-01-1-0130, PC051308 from Congressionally Directed Medical Research Programs, grant BCTR0706983 from The Susan G. Komen Breast Cancer Foundation, and grant 2008-02 from The US Military Cancer Institute and Henry M. Jackson Foundation to Dr. Yan-gao Man, and by grant 2006CB910505 from The Ministry of Chinese Science and Technology Department to Drs. Xichen Zhang and Yan-gao Man). 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 2250.

Specific overexpression of cyclin E·CDK2 in early preinvasive and primary breast tumors in female ACI rats induced by estrogen.

Overexpressed Aurora A, amplified centrosomes, and aneuploidy are salient features of estrogen-induced mammary preinvasive lesions and tumors in female August--Copenhagen Irish (ACI) rats. Intimately involved in these events are cyclins and their associated cyclin-dependent kinase (CDK) partners. Cyclin E1·CDK2 overexpression plays an important dual role in late G1/S phase of the cell cycle in cancer cells. It increases DNA replication providing growth advantage to cancer cells and facilitates aberrant centrosome duplication, generating chromosomal instability and aneuploidy leading to tumor development. Presented herein, a 24.0- and 45.0-fold elevation in cyclin E1 and CDK2 was found in 17β-estradiol (E(2))-induced ACI rat mammary tumors (MTs), respectively. Cyclin E·CDK2 positive staining was confined to the large round cells found within focal dysplasias, ductal carcinomas in situ, and invasive MTs. Co-immunoprecipitation and in vitro kinase activity of these tumors revealed that these cell cycle entities are functional. When mammary tissue derived from untreated normal, E(2)-induced hyperplasia and primary tumors were normalized to cyclin E1 levels, low molecular weight (LMW) cyclin E1 forms (33- and 45-kDa) were detected in all of these tissue groups. Moreover, increasing concentrations of protease inhibitor in tissue lysates resulted in a marked reduction of LMW forms, indicating that the presence of cyclin E1 LMW forms can be markedly reduced. Significant increases in cyclin E1 mRNA (2.1-fold) were detected in primary ACI rat E(2)-induced breast tumors, and quantitative real-time polymerase chain reaction revealed a 20% amplification of the cyclin E1 gene (CCNE1). Collectively, these results support the involvement of cyclin E1·CDK2 in centrosome overduplication during each stage of E(2)-induced mammary tumorigenesis.

Distinct populations of metastases-enabling myeloid cells expand in the liver of mice harboring invasive and preinvasive intra-abdominal tumor

The liver is the most common site of adenocarcinoma metastases, even in patients who initially present with early disease. We postulated that immune-suppressive cells in the liver of tumor-bearing hosts inhibit anti-tumor T cells, thereby accelerating the growth of liver metastases. Using models of early preinvasive pancreatic neoplasia and advanced colorectal cancer, aims of this study were to determine immune phenotype, stimulus for recruitment, inhibitory effects, and tumor-enabling function of immune-suppressive cells in the liver of tumor-bearing hosts. We found that in mice with intra-abdominal malignancies, two distinct CD11b(+)Gr1(+) populations with divergent phenotypic and functional properties accumulate in the liver, becoming the dominant hepatic leukocytes. Their expansion is contingent on tumor expression of KC. These cells are distinct from CD11b(+)Gr1(+) populations in other tissues of tumor-bearing hosts in terms of cellular phenotype and cytokine and chemokine profile. Liver CD11b(+)Gr1(+) cells are highly suppressive of T cell activation, proliferation, and cytotoxicity and induce the development of Tregs. Moreover, liver myeloid-derived suppressor cells accelerate the development of hepatic metastases by inactivation of cytotoxic T cells. These findings may explain the propensity of patients with intra-abdominal cancers to develop liver metastases and suggest a promising target for experimental therapeutics.

Mutant PIK3CA Is Detected in Both Pre-Invasive and Recurrent Breast Cancer.

Abstract BackgroundWe identified somatic PIK3CA mutations in 32.5% of 590 primary invasive breast cancers (BC) (manuscript in press: Clinical Cancer Research). Detected by massARRAY genotyping, PIK3CA mutations significantly associate with favorable clinicopathologic features and improved clinical outcome, including overall and breast cancer-specific survival. Given the strong association between PIK3CA mutations and hormone receptor (HR) positivity, one hypothesis is that PIK3CA mutations 'drive' HR positive BC and will be detected in pre-invasive breast tumors. As PIK3CA mutations offer a protective effect in BC, it has not been determined whether PIK3CA mutations are selected for in disease progression or whether additional collaborating mutations are required.MethodsTo determine the concordance of PIK3CA mutations and assess the acquisition of additional oncogene mutations, available matched tissue samples, from the previous database, were procured and underwent massARRAY genotyping (n = 83). Two mm cores were macro-dissected from matched formalin-fixed, paraffin embedded tissue, including normal breast tissue, benign lymph nodes (LN), ductal carcinoma-in-situ (DCIS), regional LN metastases (mets), and distant mets. MassARRAY (Sequenom) genotyping was performed on native DNA to identify rare and hotspot PIK3CA mutations, as well as AKT1 (E17K), RAS, and RET mutations.ResultsConcordance of PIK3CA mutations is noted between primary BC and DCIS, except for one rare PIK3CA mutation (Q546R) not detected in DCIS (4/5). No PIK3CA mutations are detected in normal breast tissue (0/8) or benign LN (0/6). Rare and hotspot PIK3CA mutations in primary BC are detected in 87.5% (7/8) matched regional LN met and 80 % (4/5) distant mets. For the patient with the disconcordant regional LN met, the rare PIK3CA mutation (H1047L) identified in primary BC is also present in the distant met site. Notably, for the single disconcordant PIK3CA met site, a rare PIK3CA mutation (E545A) is detected in a primary tumor, regional LN met, and bone met and is absent in a second bone met, in which a KRAS (G12C) mutation has been identified. One concomitant hotspot PIK3CA (E542K)/KRAS (G12C) mutation is present in both primary BC and paired DCIS. Complete concordance of AKT1 (E17K) mutations has been identified between in DCIS, primary BC, regional LN met, and distant mets (n=4).ConclusionsWe recently defined the positive prognostic significance of PIK3CA mutations in breast cancer. PIK3CA and AKT1 (E17K) mutations are early events in breast cancer, occurring in pre-invasive tumors. Despite the protective effect of PIK3CA mutations on clinical outcome, they persist and are selected for in disease progression, as they are detected in regional LN and distant mets. Complete concordance is identified between hotspot PIK3CA mutated primary BC and matched tumors samples, suggesting that PIK3CA mutations with higher oncogenic potency are maintained in tumor progression. These findings support that targeting the PI3K pathway may assist in tailoring therapy to appropriate patient populations. We are expanding matched tissue collection from a separate patient cohort to further assess genomic and functional genomic change with disease progression. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5164.

Serum protein signature may improve detection of ductal carcinoma in situ of the breast

Ductal carcinoma in situ (DCIS) of the breast is part of a spectrum of preinvasive lesions that originate within normal breast tissue and progress to invasive breast cancer. The detection of DCIS is important for the reduction of mortality from breast cancer, but the diagnosis of preinvasive breast tumors is hampered by the lack of an adequate detection method. To identify the changes in protein expression during the initial stage of tumorigenesis and to identify the presence of new DCIS markers, we analysed serum from 60 patients with breast cancer and 60 normal controls using mass spectrometry. A 23-protein index was generated that correctly distinguishes the DCIS and control groups with sensitivities and specificities in excess of 80% in two independent cohorts. Two candidate peptides were purified and identified as platelet factor 4 (PF-4) and complement C3a(desArg) anaphylatoxin (C3a(desArg)) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In an independent serum set of 165 patients, PF-4 and C3a(desArg) were significantly upregulated in DCIS compared with non-cancerous controls, as validated using western blot and enzyme-linked immunosorbent assay. We conclude that our serum protein-based test, used in conjunction with image-based screening practices, could improve the sensitivity and specificity of breast cancer detection.

Loss of RhoB Expression Promotes Migration and Invasion of Human Bronchial Cells Via Activation of AKT1

Lung cancer is the leading cause of cancer-related death worldwide, mainly due to its highly metastatic properties. Previously, we reported an inverse correlation between RhoB expression and the progression of the lung cancer, occurring between preinvasive and invasive tumors. Herein, we mimicked the loss of RhoB observed throughout lung oncogenesis with RNA interference in nontumoral bronchial cell lines and analyzed the consequences on both cell transformation and invasion. Down-regulation of RhoB did not modify the cell growth properties but did promote migration and invasiveness. Furthermore, RhoB depletion was accompanied by modifications of actin and cell adhesion. The specific activation of the Akt1 isoform and Rac1 was found to be critical for this RhoB-mediated regulation of migration. Lastly, we showed that RhoB down-regulation consecutive to K-RasV12 cell transformation is critical for cell motility but not for cell proliferation. We propose that RhoB loss during lung cancer progression relates to the acquisition of invasiveness mediated by the phosphatidylinositol 3-kinase (PI3K)/AKT and Rac1 pathways rather than to tumor initiation.

Phosphorylation of the src Epithelial Substrate Trask Is Tightly Regulated in Normal Epithelia but Widespread in Many Human Epithelial Cancers

The frequently elevated activities of the c-src and c-yes products in human epithelial tumors suggest that these activated tyrosine kinases have tumorigenic functions analogous to the v-src and v-yes oncogene products. Studies of v-src-transformed fibroblasts have identified many of the effectors of this potent oncogene; however, because c-src and c-yes lack the mutational and promiscuous activities of their retroviral oncogene homologues, their presumptive tumorigenic functions in human epithelial tumors are more subtle, less well-defined, and await identification of possible effectors more directly relevant to epithelial cells. We recently identified a transmembrane glycoprotein named Trask that is expressed in epithelial tissues but not fibroblasts and is phosphorylated by SRC kinases in mitotic epithelial cells. In this study, we have surveyed the expression and phosphorylation of Trask in many human epithelial cancer cell lines and surgical tissues and tumors. Trask is widely expressed in human epithelial tissues, but its phosphorylation is tightly regulated and restricted to detached mitotic cells or cells undergoing physiologic shedding. However, abberant Trask phosphorylation is seen in many epithelial tumors from all stages including preinvasive, invasive, and metastatic tumors. Trask phosphorylation requires SRC kinases, and is also aberrantly hyperphosphorylated in the SRC-activated PyMT mouse epithelial tumors and dephosphorylated by the SRC inhibitor treatment of these tumors. The widespread phosphorylation of Trask in many human epithlelial cancers identifies a new potential effector of SRC kinases in human epithelial tumorigenesis.

Activation of EGFR signaling in CD44 positive cells promotes aggressive behavior and progression of breast cancer.

Abstract Abstract #5056 Ductal carcinoma in situ (DCIS) of the breast is a pre-invasive tumor stage with a hallmark of filling the breast ducts with tumor cells. At present, the mechanism of progression of DCIS to invasive breast cancer is poorly defined. Understanding the molecular events controlling this process should allow for the development of biomarkers for personalized medicine. Increasing evidence suggests that breast cancer is initiated from a few transformed cells that are capable of self-renewal, which is the feature of stem cells. The presence of cancer stem cells in the CD44+/CD24- cells is demonstrated in human breast cancer tissues. We found that 3 to 4% of cells in a human breast cancer cell line, MCF-10DCIS.com (MCF-10DCIS), strongly express CD44. Although MCF-10DCIS cells give rise to DCIS lesions at the early stage which progresses to invasive tumors at a later stage after injection into nude mice, injection of 500 to 5000 of the CD44+ DCIS cells produced predominantly invasive tumors in a short time. However, the CD44 - cells did not produce any tumors even at 100 fold higher cell numbers. We have previously shown that EGFR activated HIF-1α upregulates the anti-apoptotic protein survivin. Our current results show that EGFR-activated HIF-1α plays an important role in the aggressive behavior of CD44+ cells and promotes DCIS progression. Several studies demonstrated that HIF-1α also increases the levels of the genes related to invasion and metastasis, such as uPAR and CXCR4. Histological examination of MCF-10DCIS tumor xenografts shows high levels of CD44, EGFR, HIF-1α and survivin are co-localized at the macroinvasive areas and basal layer of DCIS lesions. Furthermore, invaisve tumor cells are heterogeneous with a high percentage of EGFR+, CD44+, and Her-2 - cells and a low percentage of CD44+, EGFR- and Her-2 + cells. Therefore, this orthotopic model provides an excellent tumor model for identifying biomarkers that promote DCIS invasion. Recent studies have shown that breast cancers with a triple negative phenotype (ER, PR- and Her-2-) are very aggressive and significant less numbers of the cancer patients are found in the DCIS stage compared to other breast cancer types. A high percentage of triple negative cancer tissues also expresses EGFR. We found that CD44 and survivin are expressed in 60 to 70% of the triple negative invasive cancer and in 100% of triple negative DCIS tissues. CD44 and EGFR were detected in triple negative DCIS tumor cells located at the luminal, basal and microinvasive DCIS areas, while in the ER+ DCIS type, CD44 and EGFR are found in the tumor cells at the basal layer and microinvasive foci, suggesting the role of CD44 +, EGFR and survivin expressing tumor cells in progression of DCIS to invasive breast cancer. Since MCF-10DCIS cells are negative for ER and PR, this tumor model provides a great opportunity to investigate signal pathways for triple negative breast cancer as well as the development of novel therapeutics for the treatment of this aggressive cancer type. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5056.

Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma

IntroductionDuctal 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.MethodsForty-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.ResultsAmong 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.ConclusionsWe 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.

Real-time imaging reveals that noninvasive mammary epithelial acini can contain motile cells

To determine how extracellular signal–regulated kinases (ERK) 1/2 promote mammary tumorigenesis, we examined the real-time behavior of cells in an organotypic culture of the mammary glandular epithelium. Inducible activation of ERK1/2 in mature acini elicits cell motility and disrupts epithelial architecture in a manner that is reminiscent of ductal carcinoma in situ; however, motile cells do not invade through the basement membrane and branching morphogenesis does not take place. ERK1/2-induced motility causes cells to move both within the cell monolayer that contacts the basement membrane surrounding the acinus and through the luminal space of the acinus. E-cadherin expression is reduced after ERK1/2 activation, but motility does not involve an epithelial–mesenchymal transition. Cell motility and the disruption of epithelial architecture require a Rho kinase– and myosin light chain kinase–dependent increase in the phosphorylation of myosin light chain 2. Our results identify a new mechanism for the disruption of architecture in epithelial acini and suggest that ERK1/2 can promote noninvasive motility in preinvasive mammary tumors.

Focal degeneration of basal cells and the resultant auto-immunoreactions: A novel mechanism for prostate tumor progression and invasion

The development of human prostate cancer is believed to be a multistep process, progressing sequentially from normal, to hyperplasia, to prostatic intraepithelial neoplasia (PIN), and to invasive and metastatic lesions. High grade PIN has been generally considered as the direct precursor of invasive lesions, and the progression of PIN is believed to be triggered primarily, if not solely, by the overproduction of proteolytic enzymes predominately by cancer cells, which result in the degradation of the basement membrane. These theories, however, are hard to reconcile with two main facts: (1) only about 30% untreated PIN progress to invasive stage, while none of the current approaches could accurately identify the specific PIN or individuals at greater risk for progression, and (2) results from recent world-wide clinical trials with a wide variety of proteolytic enzyme inhibitors have been very disappointing, casting doubt on the validity of the proteolytic enzyme theory. Since over 90% of prostate cancer-related deaths result from invasion-related illness and the incidence of PIN could be up to 16.5-25% in routine or ultrasound guided prostate biopsy, there is an urgent need to uncover the intrinsic mechanism of prostate tumor invasion. Promoted by the facts that the basal cell population is the source of several tumor suppressors and the absence of the basal cell layer is the most distinct feature of invasive lesions, our recent studies have intended to identify the early alterations of basal cell layers and their impact on tumor invasion using multidisciplinary approaches. Our studies revealed that a subset of pre-invasive tumors contained focal disruptions (the absence of basal cells resulting in a gap greater than the combined size of at least three epithelial cells) in surrounding basal cell layers. Compared to their non-disrupted counterparts, focally disrupted basal cell layers had several unique features: (1) significantly lower proliferation; (2) significantly lower p63 expression; (3) significantly higher apoptosis; and (4) significantly higher leukocyte infiltration and stromal reactions. Compared to their counterparts distant from focal disruptions or overlying non-disrupted basal cell layers, epithelial cells overlying focal basal cell layer disruptions showed the following unique features: (1) significantly higher proliferation; (2) significantly higher expression of cell cycle control-, cell growth-, and stem cell-related genes; and (3) physical continuity with adjacent invasive lesions. Together, these findings suggest that focal basal cell layer disruptions could substantially impact the molecular profile and biological presentations of the overlying epithelial cells. Based on these and other findings, we have proposed that prostate tumor invasion is triggered by a localized degeneration of aged or injured basal cells and the resultant auto-immunoreactions. Our hypothesized steps for prostate tumor invasion include the following: (1) due to inherited or environmental factors, some patients contained cell cycle control- and renewal-related defects in the basal cell population that cause elevated basal cell degenerations; (2) the degradation products of degenerated basal cells or diffusible molecules of the overlying epithelial cells attract leukocyte infiltration; (3) leukocytes discharge their digestive enzymes upon the direct physical contact, resulting in a focal disruption in the basal cell layer, which leads to several focal alterations: (a) a focal loss of tumor suppressors and paracrine inhibitory function; (b) a focal increase of the permeability for growth-required nutrients and oxygen; (c) a focal increase of growth factors; (d) direct physical contact between epithelial and stromal cells; and (e) the exposure of the overlying epithelial cells directly to the stromal tissue fluid. These alterations individually or collectively stimulate or favor a clonal proliferation and stromal invasion of tumor progenitor or stem cells. Our hypothesis differs from the traditional theories in several aspects, including the triggering factor for the initiation of tumor invasion, the stage of tumor invasion, the cellular origin of invasive lesions, the significance of immunoreactive and stromal cells, and the potential approaches for early detection, treatment, and prevention of invasion. Our hypothesis represents a novel in vivo model as to the cellular mechanism leading to prostate tumor invasion. If confirmed, it could lead to a new direction to search for more effective approaches to combat prostate cancer. It could also have an immediate impact on patient care through improved pathologic evaluation of prostate tumor biopsies. More importantly, our hypothesis might be applicable, and significantly impact the detection, treatment, and prevention of other epithelium-derived tumors.

Distinct Mitotic Segregation Errors Mediate Chromosomal Instability in Aggressive Urothelial Cancers

Chromosomal instability (CIN) is believed to have an important role in the pathogenesis of urothelial cancer (UC). The aim of this study was to evaluate whether disturbances of mitotic segregation contribute to CIN in UC, if these processes have any effect on the course of disease, and how deregulation of these mechanisms affects tumor cell growth. We developed molecular cytogenetic methods to classify mitotic segregation abnormalities in a panel of UC cell lines. Mitotic instabilities were then scored in biopsies from 52 UC patients and compared with the outcome of tumor disease. Finally, UC cells were exposed in vitro to a telomerase inhibitor to assess how this affects mitotic stability and cell proliferation. Three distinct chromosome segregation abnormalities were identified: (a) telomere dysfunction, which triggers structural rearrangements and loss of chromosomes through anaphase bridging; (b) sister chromatid nondisjunction, which generates discrete chromosomal copy number variations; and (c) supernumerary centrosomes, which cause dramatic shifts in chromosome copy number through multipolar cell division. Chromosome segregation errors were already present in preinvasive tumors and a high rate mitotic instability was an independent predictor of poor survival. However, induction of even higher levels of the same segregation abnormalities in UC cells by telomerase inhibition in vitro led to reduced tumor cell proliferation and clonogenic survival. Several distinct chromosome segregation errors contribute to CIN in UC, and the rate of such mitotic errors has a significant effect on the clinical course. Efficient tumor cell proliferation may depend on the tight endogenous control of these processes.

Identification of Cyclin D1– and Estrogen-Regulated Genes Contributing to Breast Carcinogenesis and Progression

Tumors can become lethal when they progress from preinvasive lesions to invasive carcinomas. Here, we identify candidate tumor progression genes using gene array analysis of preinvasive and invasive tumors from mice, which were then evaluated in human cancers. Immediate early response protein IEX-1, small stress protein 1 (HSPB8), and tumor necrosis factor-associated factor-interacting protein mRNAs displayed higher expression levels in invasive lesions than in preinvasive lesions using samples obtained by laser capture microdissection (LCM) from transgenic erbB2, ras, and cyclin D1 mice. LCM-isolated tissues from patient-matched normal, ductal carcinoma in situ, and invasive ductal carcinoma revealed similar increased expression in invasive human cancers compared with preinvasive and normal samples. These genes induced anchorage independence, increased cell proliferation, and protected against apoptosis, singly or in collaboration with erbB2. Surprisingly, they were all up-regulated by 17beta-estradiol and cyclin D1, and cyclin D1 overexpression increased p300/CBP binding to their promoters, supporting the model that cyclin D1-estrogen receptor (ER) coactivator interactions may be important to its role in ER-positive breast cancer. Additionally, an irreversible dual kinase inhibitor of ErbB signaling inhibited expression of the same genes. The up-regulation of genes contributing to increased invasiveness of ER-positive cancers offers a novel explanation for the contribution of cyclin D1 to a worse prognosis in ER-positive cancers. As targets of estrogen, cyclin D1, and erbB2 signaling, these candidates offer insights into the nature of the second events involved in breast cancer progression, regulatory events contributing to invasion, and potential targets of combined inhibition of hormone and growth factor signaling pathways.

Transition from Preinvasive Carcinoma In Situ to Seminoma Is Accompanied by a Reduction of Connexin 43 Expression in Sertoli Cells and Germ Cells

Carcinoma in situ (CIS) represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap functional proteins, connexin 43 (Cx43) represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semi quantitative reverse transcription-polymerase chain reaction (RT-PCR) from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and germ cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development.

Fluorodeoxyglucose Uptake Correlates with the Growth Pattern of Small Peripheral Pulmonary Adenocarcinoma

Noguchi and colleagues reported that the growth pattern of small-sized adenocarcinoma was related to the vascular involvement and the prognosis of the patient. Noguchi's type A/B tumors had no lymph node metastasis, rare vascular involvement, and an excellent prognosis, which meant that Noguchi's type A/B tumors were preinvasive tumors of the peripheral type. Although Noguchi's classification was usually determined based on resected specimens, it would be useful to make a decision about the therapeutic strategy if the classification could be determined preoperatively based on the fluorodeoxyglucose (FDG) uptake. The FDG uptake in 61 pulmonary adenocarcinomas measuring 3 cm or smaller in diameter was compared with the mediastinal uptake and was classified into five grades. The relationship between the FDG uptake and Noguchi's classification (A to F) was analyzed. The FDG uptake was significantly lower in Noguchi's type A/B tumors than in type C or in type D/E/F. Eleven of 12 tumors (92%) with no increased or a weak FDG uptake were classified as type A/B, whereas 32 of 33 tumors (97%) with a strong or very strong FDG uptake were classified as type C-F. The FDG uptake is helpful for making an accurate diagnosis of Noguchi's classification preoperatively in patients with pulmonary adenocarcinoma.

Int7G24A Variant of Transforming Growth Factor-β Receptor Type I Is Associated with Invasive Breast Cancer

The transforming growth factor-beta (TGF-beta) signaling pathway has been frequently implicated in breast cancer. An intronic variant (Int7G24A) of TGF-beta receptor type I (TGFBR1) is associated with kidney and bladder cancers in our recent study. We hypothesize that this germline variant may be involved in development and progression of breast cancer. Case-control studies were designed from archived paraffin-embedded tissue specimens from the same geographic area with a homogenous ethnic population. We analyzed 223 patients (25 with preinvasive tumors and 198 with invasive and metastatic breast cancers) and 153 noncancer controls. The Int7G24A was identified by PCR-RFLP. Another germline deletion (TGFBR1*6A) and somatic mutations in the TGFBR1 were also analyzed by PCR and single-strand conformational polymorphism. The Int7G24A allele was evident in 32% of patients with preinvasive neoplasms and 48% of patients with invasive breast cancers compared with 26% controls (P = 0.00008). In addition, 11 (5.6%) homozygous Int7G24A carriers were found in patients with invasive breast cancers, whereas only 3 (2%) homozygous carriers were found in the control group. The TGFBR1*6A allele was not significantly associated with breast cancer patients and only one somatic mutation was found in 71 breast cancers. These data suggest that the germline Int7G24A variant may represent a risk factor for invasive breast cancer and a marker for breast cancer progression. A separate study with a larger sample size is warranted to validate the association of the Int7G24A with human breast cancer.

Microsatellite instability analysis on epithelial ovarian tumor

Objective：Some genetic alterations are involved in ovarian carcinogenesis. Since some benign and borderline tumors may represent early steps in ovarian carcinogenesis, analysis of precursor lesions could provide evidence that an accumulation of genetic events is required in order for normal ovarian epithelium to generate benign, borderline, malignant tumors. Few pre-invasive ovarian tumors have been studied so far. Methods：60 cases of ovarian epithelial tumors, including benign, borderline, and malignant tumors, were analyzed for microsatellite instability (MSI) by gel analysis of paired germ line and tumor DNA. PCR amplification was performed using the panel of 5 microsatellite markers recommended by the NCI (BAT25, BAT26, D2S123, D5S346, D17S250) and 6 additional markers (D1S160, D1S162, D7S522, D11S860, D17S855, D17S932). Results：In this study, D2S123 and D5S346 were the most frequently altered markers in malignant ovarian tumors and D11S860 locus showed MSI in 4 adenomas, 4 boderline tomors, and 10 malignant tumors. Other markers displayed instability with only one or two tumors showing MSI. On the basis of NCI criteria, most benign tumors demonstrat microsatellite stable (MSS). In the borderline tumors, 10 tumors revealed MSS, 8 tumors had low-frequency MSI (MSI-L), and two tumors had high frequency MSI (MSI-H). In the malignant tumors, 6 tumors revealed MSS, 2 tumors had MSI-L, and 12 tumors had high MSI-H. Conclusion：According to the results, MSL-H is more frequently seen in the malignant tumors. D2S123 and D5S346 were the most frequently altered markers in the malignant tumors, and D11S860 locus may be involved in early step of carcinogenesis.