Showing papers by "Paul J. van Diest published in 2005"

Twist overexpression induces in vivo angiogenesis and correlates with chromosomal instability in breast cancer.

Abstract: Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF in vivo were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability in vivo than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype.

The origin of vimentin expression in invasive breast cancer: epithelial-mesenchymal transition, myoepithelial histogenesis or histogenesis from progenitor cells with bilinear differentiation potential?

Abstract: Vimentin expression is a rather rare finding in invasive breast cancer, and is associated with high tumour invasiveness and chemoresistance. It is currently explained by two different biological theories: direct histogenetic derivation from myoepithelial cells, and epithelial-mesenchymal transition (EMT) reflecting the end-stage of breast cancer dedifferentiation. In this study we aimed to obtain further insights into the biological hallmarks of these vimentin-expressing breast cancers. We applied immunohistochemistry for vimentin and 15 other differentiation markers to a series of 364 invasive breast cancer cases, using tissue microarray technology. 7.7% of all tumours expressed vimentin. Almost all of these cases (19/21) were Grade 3 invasive ductal carcinomas, and the majority (13/21) of these were associated with a ductal in situ component. Vimentin expression was also seen in the respective in situ components and correlated positively with the expression of SMA, CD10, CK 5, p53, Mib-1 and EGFR. A negative correlation was seen for the expression of CK 8/18 and the oestrogen receptor. Vimentin-expressing carcinomas revealed a significantly higher average absolute number of cytogenetic alterations per case, but a significantly lower frequency of chromosome 16q losses compared to vimentin-negative cases. Our present results demonstrate that, despite analogies between vimentin-positive breast cancers and myoepithelial cells in their expression of differentiation-related proteins, neither myoepithelial histogenesis nor EMT can exclusively explain the biology of these distinct tumours. This is mainly supported by the significantly higher incidence of vimentin-expressing breast cancers compared to any other myoepithelial breast tumours and the fact that vimentin is already observed in ductal in situ components. We therefore propose the alternative hypothesis that vimentin-expressing breast carcinomas may derive from breast progenitor cells with bilinear (glandular and myoepithelial) differentiation potential.

The molecular genetics and morphometry-based endometrial intraepithelial neoplasia classification system predicts disease progression in endometrial hyperplasia more accurately than the 1994 world health organization classification system

Abstract: BACKGROUND. The objective of this study was to compare the accuracy of disease progression prediction of the molecular genetics and morphometry-based Endometrial Intraepithelial Neoplasia (EIN) and World Health Organization 1994 (WHO94) classification systems in patients with endometrial hyperplasias. METHODS. A multicenter, multivariate analysis was conducted on 477 patients with endometrial hyperplasia who were required to have a 1-year minimum disease-free interval from the time of the index biopsy (1-18 years of follow-up). The results from that analysis were compared with the results from 197 patients who had < 1 year of follow-up. RESULTS. Twenty-four of 477 hyperplasias (5.0%) progressed to malignant disease over an average of 4 years (maximum, 10 years). According to the WHO94 classification, 16 of 123 atypical hyperplasias (13%) and 8 of 354 nonatypical hyperplasias (2.3%) progressed (hazard ratio [HR] = 7). Twenty-two of 118 EINs (19%) and 2 of 359 non-EINs (0.6%) progressed (HR = 45). EIN was prognostic within each WHO94 subcategory. Progression rates were 3% in simple hyperplasias, 22% in complex hyperplasias, 17% in simple atypical hyperplasias, and 38% in complex atypical hyperplasias with EIN, compared with progression rates of 0.0-2.0% in all hyperplasias if EIN was absent. EIN detected precancerous lesions (sensitivity, 92%) better than WHO94 atypical hyperplasias collectively (67%) or complex atypical hyperplasias alone (46%). In a Cox regression analysis, EIN was the strongest prognostic index of future endometrial carcinoma. The same was true for patients with < 1 year of follow-up (HR for EIN, atypical hyperplasia, and complex atypical hyperplasia: 58, 7, and 8, respectively). CONCLUSIONS. The EIN classification system predicted disease progression more accurately than the WHO94 classification and identified many women with benign changes that would have been regarded as high risk according to the WHO94 classification system.

HIF1-alpha overexpression indicates a good prognosis in early stage squamous cell carcinomas of the oral floor

Abstract: Hypoxia-inducible factor 1 (HIF-1) is a transcription factor, which plays a central role in biologic processes under hypoxic conditions, especially concerning tumour angiogenesis. HIF-1α is the relevant, oxygen-dependent subunit and its overexpression has been associated with a poor prognosis in a variety of malignant tumours. Therefore, HIF-1α expression in early stage oral carcinomas was evaluated in relation to established clinico-pathological features in order to determine its value as a prognostic marker. 85 patients with histologically proven surgically treated T1/2 squamous cell carcinoma (SCC) of the oral floor were eligible for the study. Tumor specimens were investigated by means of tissue micro arrays (TMAs) and immunohistochemistry for the expression of HIF-1. Correlations between clinical features and the expression of HIF-1 were evaluated by Kaplan-Meier curves, log-rank tests and multivariate Cox regression analysis. HIF-1α was frequently overexpressed in a probably non-hypoxia related fashion. The expression of HIF-1α was related with a significantly improved 5-year survival rate (p < 0.01) and a significantly increased disease free period (p = 0.01) independent from nodal status and tumour size. In primary node negative T1/T2 SCC of the oral floor, absence of HIF-1α expression specified a subgroup of high-risk patients (p < 0.05). HIF-1α overexpression is an indicator of favourable prognosis in T1 and T2 SCC of the oral floor. Node negative patients lacking HIF-1α expression may therefore be considered for adjuvant radiotherapy.

Ischemia/reperfusion accelerates the outgrowth of hepatic micrometastases in a highly standardized murine model

Abstract: Proc Amer Assoc Cancer Res, Volume 46, 2005 5659 Mortality in colorectal cancer is associated with the development of liver metastases. Surgical removal of these tumors is the only hope for cure, but recurrence is common. During hepatic surgery, ischemia/reperfusion (I/R) injury of the liver often occurs due to vascular clamping. The adverse effects of I/R on hepatocellular damage and postoperative liver function are well documented. The influence on the outgrowth of residual tumor deposits is unknown. The aim of this study was to investigate the effect of hepatic I/R on the outgrowth of pre-established colorectal micrometastases in a highly standardized murine model and to evaluate the putative protective effect of alternative clamping methods. Five days following intrasplenic injection with C26 colon carcinoma cells the vascular structures to the left lobe were clamped for 45 minutes under hemodynamically stable conditions. Markers of oxidative stress (GSH), hepatocellular damage (ALT and AST) and tumor growth were assessed over time. Next, we analyzed the effect of ischemic preconditioning, intermittent clamping and selective clamping of the portal vein on hepatocellular damage and tumor growth. I/R induced oxidative stress and hepatocellular damage as measured by decreased liver GSH, a marked increase in liver enzymes and the presence of hepatic necrosis covering 14% of the liver parenchyma. The outgrowth of micrometastases in occluded liver lobes was accelerated 5-6 fold when compared to non-occluded liver lobes and was associated with the presence of necrotic areas. Both early and late hepatocellular damage were prevented by occluding blood flow intermittently or by portal clamping as indicated by a 96% reduction in liver enzymes and by the absence of necrosis. In addition, accelerated tumor growth was completely prevented by both methods. In contrast, although early hepatocellular damage was largely prevented (87%), ischemic preconditioning reduced hepatocellular necrosis only by 50% and failed to protect against accelerated tumor growth. Our results identify I/R as a strong stimulus of recurrent intrahepatic tumor growth and indicate that protection against I/R-induced tissue necrosis cross-protects against this phenomenon. ![Figure][1] [1]: pending:yes

Prospective Multicenter Validation of the Independent Prognostic Value of the Mitotic Activity Index in Lymph Node–Negative Breast Cancer Patients Younger Than 55 Years

Abstract: Purpose To validate the independent strong prognostic value of mitotic activity index (MAI) in lymph node (LN) –negative invasive breast cancer patients younger than 55 years in a nationwide multicenter prospective study. Patients and Methods Analysis of routinely assessed MAI and other prognosticators in 516 patients (median follow-up, 118 months; range, 8 to 185 months), without systemic adjuvant therapy or previous malignancies. Results Distant metastases occurred in 127 patients (24.6%); 90 (17.4%) died as a result of metastases. MAI (< 10, ≥ 10) showed strong association with recurrence (hazard ratio [HR], 3.12; 95% CI, 2.17 to 4.50; P ≤ .0001) and mortality (HR, 4.42; 95% CI, 2.79 to 7.01; P < .0001). The absolute difference in 10-year Kaplan-Meier estimates of time to distant recurrence as well as survival was 22% between MAI less than 10 versus ≥ 10. This effect was independent of age, estrogen receptor (ER) status, and tumor diameter (which were significant prognosticators). In multivariate analy...

Development of 3D Chromatin Texture Analysis Using Confocal Laser Scanning Microscopy

Abstract: Introduction: Analysis of nuclear texture features as a measure of nuclear chromatin changes has been proven to be useful when measured on thin (5–6 μm) tissue sections using conventional 2D bright field microscopy. The drawback of this approach is that most nuclei are not intact because of those thin sections. Confocal laser scanning microscopy (CLSM) allows measurements of texture in 3D reconstructed nuclei. The aim of this study was to develop 3D texture features that quantitatively describe changes in chromatin architecture associated with malignancy using CLSM images. Methods: Thirty-five features thoughtfully chosen from 4 categories of 3D texture features (discrete texture features, Markovian features, fractal features, grey value distribution features) were selected and tested for invariance properties (rotation and scaling) using artificial images with a known grey value distribution. The discriminative power of the 3D texture features was tested on artificially constructed benign and malignant 3D nuclei with increasing nucleolar size and advancing chromatin margination towards the periphery of the nucleus. As a clinical proof of principle, the discriminative power of the texture features was assessed on 10 benign and 10 malignant human prostate nuclei, evaluating also whether there was more texture information in 3D whole nuclei compared to a single 2D plane from the middle of the nucleus. Results: All texture features showed the expected invariance properties. Almost all features were sensitive to variations in the nucleolar size and to the degree of margination of chromatin. Fourteen texture features from different categories had high discriminative power for separating the benign and malignant nuclei. The discrete texture features performed less than expected. There was more information on nuclear texture in 3D than in 2D. Conclusion: A set of 35 3D nuclear texture features was used successfully to assess nuclear chromatin patterns in 3D images obtained by confocal laser scanning microscopy, and as a proof of principle we showed that these features may be clinically useful for analysis of prostate neoplasia.

Dual effect of Krasd12 knockdown on tumorigenesis : increased immune-mediated tumor clearance and abrogation of tumor malignancy

Abstract: Activating mutations in the human KRAS proto-oncogene are acquired during the earliest stages of colorectal cancer development. If mutant KRAS is to be used as a target for therapy in colorectal cancer, tumor growth should depend on its continued presence. Here, we report that stable knockdown of Kras(D12) in murine C26 colorectal cancer cells by RNA interference resulted in loss of transformed properties in vitro. The incidence of subcutaneous tumor formation was reduced by 60% and the lag time was increased sevenfold. Kras(D12)-knockdown tumors grew noninvasively and did not cause morbidity. Remarkably, some of the Kras(D12)-knockdown tumors regressed spontaneously, which rendered these mice resistant to parental C26 tumor growth. In immune-deficient hosts, the incidence of tumor formation by Kras(D12)-knockdown cells was 100%. None of these tumors regressed spontaneously. We conclude that the reduced incidence of tumor formation by Kras(D12)-knockdown cells is due to tumor cell clearance by the host immune system, but not to an intrinsic inability of these cells to grow out as tumors. Interestingly, Kras(D12) knockdown resulted in increased production of interleukin 18 (Il-18), an immune-stimulatory cytokine that has been implicated in limiting colorectal tumor formation. Thus, mutant Kras(D12) suppresses Il-18 production in colorectal tumor cells, which may contribute to evasion of the local immune system during tumor development.

Lobulitis is a frequent finding in prophylactically removed breast tissue from women at hereditary high risk of breast cancer.

Abstract: The aim of this study was to investigate closely the nature of premalignant lesions that occur in prophylactically removed breast tissue from patients at hereditary high risk of breast cancer. Breast tissues obtained from 41 patients who underwent prophylactic mastectomy (pM) because of a hereditary high risk of breast cancer and control tissues from 82 age-matched healthy controls who underwent breast reduction surgery were screened for premalignant lesions. Premalignant and malignant lesions were more frequent (p = 0.0016) in pM samples (5/41) than in controls (1/82). Interestingly, lobulitis, defined as more than 100 lymphocytes and/or plasma cells per lobule in more than one section in morphologically normal lobules, was encountered in 21 of 41 (51%) pM patients, in contrast to only 8 of 82 (10%) controls (p < 0.0001). Preliminary observations indicate a predominance of T-cells in these infiltrates, in agreement with the already known frequent presence of lymphocytic infiltration in hereditary ductal in situ and infiltrating ductal/medullary carcinomas. This novel finding implies an immune reaction to an as yet unidentified antigen frequently present in women at hereditary high risk of breast cancer, possibly as part of an early carcinogenic event.

Cyclooxygenase-2 is a target of KRASD12, which facilitates the outgrowth of murine C26 colorectal liver metastases.

Abstract: Purpose: Mutational activation of the KRAS oncogene and overexpression of cyclooxygenase-2 (COX-2) contribute to colorectal carcinoma (CRC) development, but the relationship between these two events is unclear. This study was designed to clarify that relationship and to assess the contribution of KRAS-dependent COX-2 to the seeding of CRC cells in the liver and to their outgrowth as liver metastases in an experimental mouse model. Experimental Design: The effect of RNA interference–mediated KRAS knockdown on COX-2 expression and activity was tested in murine C26 CRC cells. The contribution of KRAS-dependent COX-2 to early metastatic tumor cell seeding (by intravital microscopy) and outgrowth of metastases in the liver (by bioluminescence imaging) was studied by using parecoxib, a novel and highly selective liver-activated COX-2 inhibitor. Intratumoral cell proliferation, apoptosis, and tumor-associated angiogenesis were assessed by immunohistochemistry on liver tissue sections. Results: Stable knockdown of mutant KRASD12 in murine C26 CRC cells by RNA interference lead to a dramatic reduction of COX-2 synthesis and prostaglandin E2 production. Inhibition of host or tumor cell COX-2 activity had no effect on early metastatic cell seeding in the liver but greatly reduced intrahepatic tumor cell proliferation and the rate of liver metastasis outgrowth. COX-2 inhibition had no effect on early tumor vascularization or on tumor cell apoptosis. Conclusions: The high levels of COX-2 enzyme and prostaglandin production in C26 CRC cells are primarily caused by the presence of endogenous mutant KRASD12. Furthermore, COX-2 inhibition affects the tumoral rather than the vascular compartment during the early stages of C26 liver metastasis outgrowth.

Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast

Abstract: Introduction Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance of HIF-1α and its downstream targets in phyllodes tumors and fibroadenomas of the breast.

Progressive Deregulation of the Cell Cycle With Higher Tumor Grade in the Stroma of Breast Phyllodes Tumors

Abstract: We studied cell cycle-regulating proteins in phyllodes tumor pathogenesis by immunohistochemical analysis for Ki-67, cyclin A, cyclin D1, retinoblastoma protein (pRb), p53, p16INK4A, bcl-2, and p21waf1 in the epithelium and stroma of 40 primary (benign, 21; borderline, 8; malignant, 11) and 7 recurrent tumors of different grades. In most cases, the epithelium showed no altered expression of cell cycle regulators. Stromal overexpression of p16INK4A, p53, cyclin A, pRb, and p21waf1 correlated significantly with tumor grade. The number of altered proteins in stroma increased with higher grade and was accompanied by increased proliferation. Stromal cyclin A expression was the best separating marker between tumor grades. Correlations existed between stromal overexpression of p16NK4A and p21waf1, p16INK4A and p53, and p53 and pRb. No immunostaining differences were detected between primary tumors and recurrences. Four or more altered proteins and p53 expression in the stromal component were independent negative prognosticators for disease-free survival. The stromal component of mammary phyllodes tumors displays an increasing level of cell cycle deregulation with higher tumor grade; the epithelial compartment mostly remains inconspicuous. Several combinations of aberrantly expressed cell cycle proteins seem important in the stromal progression of phyllodes tumors. The number of stromal cell cycle aberrations and stromal p53 expression might predict clinical behavior.

Implementation of accurate and fast DNA cytometry by confocal microscopy in 3D

Abstract: Background: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. Methods: Nuclear DNA was stained in 14 μm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of ×40 or ×100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. Results: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at ×60 or ×100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. Conclusions: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.

Value of histopathologic analysis of skin excisions by GPs.

Abstract: The clinical diagnoses of skin lesions in general practice may sometimes not be very accurate. The aim of this study was to compare clinical versus final histopathological diagnosis status (benign, pre-malignant/malignant) in 4595 consecutive submissions by GPs. The final diagnosis was pre-malignant or malignant in 215 cases (4.7%). From the 4436 lesions clinically diagnosed as benign, 134 (3.0%) were pre-malignant or malignant on final histological diagnosis. From the 159 lesions clinically diagnosed as pre-malignant or malignant, 78 (49.1%) were in fact benign, and 81 (50.9%) were indeed pre-malignant or malignant on final diagnosis. The sensitivity for a malignant diagnosis was 38%, and the specificity 98%. The proportion of pre-malignancies or malignancies was 0.9% below and 9.2% above the age of 40 years. In conclusion, histopathological investigation of skin excisions by GPs yields a high percentage of unexpected pre-malignancies and malignancies. The number of misdiagnoses was age dependent, with a proportion of 1% and 9% of pre-malignancies/malignancies in patients below and above the age of 40 years, respectively. This indicates that all skin excisions by GPs must undergo routine histopathological investigation to ensure that serious malignancies are not missed.