Cecile Colpaert

Increased Angiogenesis and Lymphangiogenesis in Inflammatory versus Noninflammatory Breast Cancer by Real-Time Reverse Transcriptase-PCR Gene Expression Quantification

Purpose: Inflammatory breast cancer is a distinct and aggressive form of locally advanced breast cancer with unique clinical and pathological features. Recently, histologic evidence of intense angiogenesis was found in inflammatory breast cancer specimens. The aim of this study was to confirm the angiogenic phenotype of inflammatory breast cancer and to investigate its potential to induce lymphangiogenesis. Experimental Design: Real-time quantitative reverse transcriptase-PCR was used to measure levels of mRNA of tumor angiogenesis and lymphangiogenesis-related factors [vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, Flt-1, KDR, Flt-4, Ang-1, Ang-2, Tie-1, Tie-2, cyclooxygenase-2, fibroblast growth factor-2 (FGF-2), Egr-1, Prox-1, and LYVE-1] in tumor specimens of 16 inflammatory breast cancer and 20 noninflammatory breast cancer patients. Tissue microarray technology and immunohistochemistry were used to study differential protein expression of some of the angiogenic factors in inflammatory breast cancer and noninflammatory breast cancer. Active lymphangiogenesis was further assessed by measuring lymphatic endothelial cell proliferation. Results: Inflammatory breast cancer specimens had significantly higher mRNA expression levels than noninflammatory breast cancer specimens of the following genes: KDR (P = 0.033), Ang-1, (P = 0.0001), Tie-1 (P = 0.001), Tie-2 (P = 0.001), FGF-2 (P = 0.002), VEGF-C (P = 0.001), VEGF-D (P = 0.012), Flt-4 (P = 0.001), Prox-1 (P = 0.005), and LYVE-1 (P = 0.013). High mRNA levels of FGF-2 and cyclooxygenase-2 corresponded to increased protein expression by immunohistochemistry. Inflammatory breast cancer specimens contained significantly higher fractions of proliferating lymphatic endothelial cells than noninflammatory breast cancer specimens (P = 0.033). Conclusions: Using real-time quantitative reverse transcriptase-PCR and immunohistochemistry, we confirmed the intense angiogenic activity in inflammatory breast cancer and demonstrated the presence of active lymphangiogenesis in inflammatory breast cancer. This may help explain the high metastatic potential of inflammatory breast cancer by lymphatic and hematogenous route. Both pathways are potential targets for the treatment of inflammatory breast cancer.

Liver metastases from colorectal adenocarcinomas grow in three patterns with different angiogenesis and desmoplasia.

The liver is a highly vascularized organ which frequently hosts metastases in patients with colorectal adenocarcinomas. The hypothesis of this study is that the hypoxic drive of angiogenesis might be minimal or absent in those growing liver metastases which are capable of preserving the stromal structure, including the numerous sinusoidal blood vessels. Representative paraffin sections of liver metastases from 26 patients with colorectal adenocarcinoma were investigated. Three different growth patterns were found. In the desmoplastic and in the pushing growth patterns (42% and 46% of all metastases, respectively), the architecture of the liver parenchyma was not preserved. In the replacement growth pattern (12% of all cases), the reticulin pattern of the liver parenchyma was conserved within the metastases at the tumour–liver parenchyma interface. The endothelial cells of the blood vessels near the interface in the metastases of the replacement type did not express CD34, nor were they surrounded by alpha‐smooth muscle actin‐positive mural cells. In the desmoplastic and in the pushing growth patterns, 23% and 52% of all blood vessels in this area were covered by pericytes. The fraction of proliferating endothelial cells was low in the metastases with a desmoplastic or a replacement growth pattern (about 3%), compared with metastases with a pushing growth pattern (11%). Tumour cell apoptosis was highest in the pushing‐type metastases and was inversely correlated with microvessel density in liver metastases. The ratio of the proliferating tumour cell fraction and the proliferating endothelial cell fraction, roughly representing the degree of angiogenesis‐dependent growth, was three‐ to four‐fold higher in the replacement‐type metastases compared with the other metastases. In summary, the present study has demonstrated that liver metastases are a heterogeneous group, with different growth patterns which predict the fraction of immature blood vessels, the fraction of proliferating endothelial cells, and the fraction of apoptotic tumour cells. The replacement growth pattern expands with minimal angiogenesis by co‐opting the stroma with the sinusoidal blood vessels of the liver. Copyright

Tumor Lymphangiogenesis in Inflammatory Breast Carcinoma: A Histomorphometric Study

Purpose: At the time of diagnosis, metastatic dissemination of tumor cells via the lymphatic system has occurred in nearly all patients with inflammatory breast cancer (IBC). The objective of this study was twofold: (a) to determine which is the most suitable marker of lymph vessels in primary breast tumors and (b) to compare histomorphometric lymph vessel variables in IBC and non-IBC. Experimental Design: Serial sections of 10 IBCs and 10 non-IBCs were immunostained for D2-40, LYVE-1, podoplanin, and PROX-1. Relative lymph vessel area, lymph vessel perimeters, and counts and lymphatic endothelial cell proliferation (LECP) were then measured in D2-40/Ki-67 double-immunostained sections of 10 normal breast tissues, 29 IBCs, and 56 non-IBCs. Results: D2-40 was the most suitable antibody for staining peritumoral and intratumoral lymph vessels. D2-40-stained intratumoral lymph vessels were present in 80% of non-IBCs and 82.8% of IBCs (P = 0.76). In non-IBC, lymph vessels located in the tumor parenchyma were smaller and less numerous than those at the tumor periphery (P < 0.0001) whereas in IBC, intratumoral and peritumoral variables were not significantly different. The mean relative tumor area occupied by lymph vessels was larger in IBC than in non-IBC (P = 0.01). LECP at the tumor periphery was higher in IBC than in non-IBC: median LECP was 5.74% in IBC versus 1.83% in non-IBC (P = 0.005). Conclusions: The high LECP in IBC suggests that lymphangiogenesis contributes to the extensive lymphatic spread of IBC.

Inflammatory breast cancer shows angiogenesis with high endothelial proliferation rate and strong E-cadherin expression

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Improved understanding of the mechanisms responsible for the differences between IBC and non-IBC might provide novel therapeutic targets. We studied 35 consecutive patients with IBC, biopsied prior to the initiation of chemotherapy. Angiogenesis was evaluated by Chalkley counting and by assessment of endothelial cell proliferation (ECP) and vessel maturity. The presence of fibrin, expression of the hypoxia marker carbonic anhydrase IX (CA IX) and epithelialcadherin (E-cadherin) expression were immunohistochemically detected. The same parameters were obtained in a group of 104 non-IBC patients. Vascular density, assessed by Chalkley counting (P<0.0001), and ECP (P=0.01) were significantly higher in IBC than in non-IBC. Abundant stromal fibrin deposition was observed in 26% of IBC and in only 8% of non-IBC (P=0.02). Expression of CA IX was significantly less frequent in IBC than in non-IBC with early metastasis (P=0.047). There was a significant positive correlation between the expression of CA IX and ECP in IBC (r=0.4, P=0.03), implying that the angiogenesis is partly hypoxia driven. However, the higher ECP in IBC and the less frequent expression of CA IX in IBC vs non-IBC points at a role for other factors than hypoxia in stimulating angiogenesis. Strong, homogeneous E-cadherin expression was found at cell–cell contacts in all but two IBC cases, both in lymphovascular tumour emboli and in infiltrating tumour cells, challenging our current understanding of the metastatic process. Both the intense angiogenesis and the strong E-cadherin expression may contribute to the highly metastatic phenotype of IBC.

Preoperative breast MRI in patients with invasive lobular breast cancer

To investigate the use of MRI in preoperative characterization of invasive lobular breast cancer (ILC) and in detection of multifocal/multicentric disease. We retrospectively reviewed T1-weighted FLASH 3D precontrast and postcontrast MR images together with subtraction images of 26 women with histopathologically proven invasive lobular cancer. Two experienced radiologists described tumor patterns of ILC independently. MR findings of unifocal, multifocal, single quadrant and multiquadrant disease were correlated with results of other imaging techniques and compared with histopathological findings as gold standard. Most ILC presented on MRI as a single spiculated/irregular, inhomogeneous mass (pattern 1, n=12) or as a dominant lesion surrounded by multiple small enhancing foci (pattern 2, n=8). Multiple small enhancing foci with interconnecting enhancing strands (pattern 3) and an architectural distortion (pattern 4) were both described in three cases. There was one case of a focal area of inhomogeneous enhancement (pattern 5) and one normal MR examination (pattern 6). Unifocal and multifocal lesions were identified on MRI in four patients with normal conventional imaging. In nine women, multiple additional lesions or more extensive multiquadrant disease were correctly identified only on MRI. MRI may play an important role in the evaluation of patients with ILC, which is often difficult to diagnose on clinical examination and conventional imaging and more likely occur in multiple sites and in both breasts. However, false-negative MR findings do occur in a small percentage of ILC.

Early distant relapse in ‘node‐negative’ breast cancer patients is not predicted by occult axillary lymph node metastases, but by the features of the primary tumour

Early distant relapse occurs in a minority of node‐negative breast cancer patients. Whether this poor prognosis can be predicted by the features of the primary tumour, or by the presence of occult metastases in the ‘negative’ lymph nodes (LNs), remains a matter of debate. One hundred and four T1–2N0M0 breast carcinoma patients were divided into two groups: group 1 (44%) showing early distant relapse with a median disease‐free survival of 25 months, and group 2 (56%) showing no evidence of disease after a median follow‐up of 91.5 months. All patients had received locoregional treatment only. All tumours were evaluated for medial/lateral location, histological type, size, grade, mitotic activity, fibrotic focus, necrosis, angiogenesis, growth pattern, and lymphatic vessel permeation. The haematoxylin and eosin‐stained slides of all axillary LNs were revised and two additional levels were cut from each paraffin block for cytokeratin immunohistochemistry. In 24 patients (23%), occult metastases were found. These consisted of single cells or small clusters (SCs) in the marginal sinus in 17 patients (16%) and of larger colonies of cells in seven patients (7%). All detected metastases were smaller than 2 mm in diameter (micrometastases). There was no significant correlation between the presence of occult LN metastases (SCs or colonies) and the prognostically important features of the primary tumour. Early metastatic disease was significantly correlated with larger tumour size (p=0.02), higher histological grade (p=0.0008), mitotic activity (p<0.0001), presence of necrosis (p=0.0004), presence of fibrotic foci (p=0.0005), angiogenesis (p=0.0009), and lymphatic vessel permeation (p=0.018). Multiple logistic regression analysis showed that histological grade and the presence of a fibrotic focus were the only independent prognostic factors and that the presence of occult LN metastases was inversely correlated with early distant relapse. Prospective prognostic studies of occult LN metastases should consider the features of the primary tumour in a multivariate analysis. Copyright

The Presence of a Fibrotic Focus in Invasive Breast Carcinoma Correlates with the Expression of Carbonic Anhydrase IX and is a Marker of Hypoxia and Poor Prognosis

The value of the fibrotic focus (FF) as a marker of intra-tumoral hypoxia in invasive breast carcinoma was assessed by studying its relationship with the expression of the hypoxia-induced carbonic anhydrase IX (CA IX), angiogenesis indices and prognosis.CA IX expression was immunohistochemically detected in 2 independent study groups, totaling 184 patients, and correlated with tumor characteristics, angiogenesis related parameters and patient outcome by univariate analysis.CA IX immunostaining scores in carcinoma cells and in tumoral fibroblasts were significantly higher in expansively growing tumors (p = 0.0001 and p < 10−4, respectively), containing an FF (p = 0.0004 and p < 10−4) and showing high histological grade (p = 0.016 and p = 0.0006).Microvessel density, quantified by Chalkley counting, was correlated with CA IX expression both in the carcinoma cells and in the fibroblasts (p = 0.0076 and p = 0.0025) and with the presence and relative size of an FF (p = 0.006). The fraction of proliferating endothelial cells was positively correlated with CA IX scores in the fibroblasts (r = 0.4, p = 0.02) and with the presence of an FF (p = 0.02). CA IX scores in the fibroblasts – and to a lesser extent in the carcinoma cells – were associated with a higher relapse rate (p = 0.006) and a worse overall survival (p = 0.003). The highest CA IX immunostaining scores were found in the fibroblasts of large FF occupying more than one-third of the tumor. A large FF was associated with worse overall survival in a consecutive patient group (p = 0.01) and with shorter disease-free (p = 0.02) and overall survival (p = 0.0005) in T1-2N0 breast cancer patients.The strong association of CA IX expression with the presence of an FF shows that the latter is a marker of intra-tumoral hypoxia. FF is useful as a surrogate marker of hypoxia-driven ongoing angiogenesis and is associated with a higher relapse rate and a worse overall survival.

Validation of a tissue microarray to study differential protein expression in inflammatory and non-inflammatory breast cancer.

AbstractAims. Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with poor prognosis. The mechanisms responsible for the aggressive clinical evolution are incompletely understood. We constructed a tissue microarray (TMA) and validated its use in translational IBC research. Differential expression of proteins that might play a role in causing the IBC phenotype was studied. Methods and results. A TMA containing 34 IBC and 41 non-stage matched non-IBC tumours was constructed. Five core biopsies were taken for each IBC and three cores for each non-IBC tumour. The TMA was validated using three approaches: (1) the excellent concordance between immunohistochemical results of the initial pathological examination and the results obtained with the TMA for ER, PR and HER2/neu (κ > 0.74); (2) the known differential expression between IBC and non-IBC for four bio-markers in IBC (ER, PR, p53 and HER2/neu) was confirmed (p < 0.01); (3) the HER2/neu status using three different antibodies (CB11, TAB250 and HercepTest) was highly concordant (κ > 0.75). Furthermore, the overexpression of E-Cadherin and RhoC GTPase in IBC (p < 0.05) was confirmed. We did not find a differential expression pattern for carbonic anhydrase IX (CA IX) and EGFR. Conclusions. Using different approaches, we have validated the use of our TMA for studying differential protein expression in IBC and non-IBC. We confirm the overexpression of E-Cadherin and RhoC GTPase in IBC. The lack of differential expression for CA IX and EGFR might suggest the pathways are equally utilised in both types of breast cancer.

Increased Sentinel Lymph Node Lymphangiogenesis is Associated with Nonsentinel Axillary Lymph Node Involvement in Breast Cancer Patients with a Positive Sentinel Node

Purpose: Lymph node (LN) lymphangiogenesis has recently been shown to be important in the premetastatic niche of sentinel LNs. To study its role in the further metastatic spread of human breast cancer, we investigated the association of angiogenesis and lymphangiogenesis in sentinel LN metastases with the presence of nonsentinel LN metastases in breast cancer patients with a positive sentinel LN. Experimental Design: Angiogenesis and lymphangiogenesis—quantified as endothelial cell proliferation fraction (ECP%) and lymphatic ECP fraction (LECP%)—were assessed in sentinel LN metastases of 65 T1/T2 patients with breast cancer using CD34/Ki67 and D2-40/Ki67 immunohistochemical double stains. Correlations were analyzed between nonsentinel LN status, LECP%, and other clinicopathologic variables (number of involved sentinel LNs, size of the primary tumor and LN metastasis, presence of lymphovascular invasion in the primary tumor, and of extracapsular growth in the sentinel LN metastasis). Results: Thirty seven out of 65 patients (56.9%) had at least one involved nonsentinel LN. Size of the sentinel LN metastasis (P = 0.001), lymphovascular invasion (P = 0.02), extracapsular growth (P = 0.02), and LECP% (P = 0.01) were correlated with a positive nonsentinel LN status. The multivariate logistic regression model retained high LECP% (odds ratios = 4.2, P = 0.01) and the presence of extracapsular growth (odds ratios = 3.38, P = 0.04) as independently associated with the presence of nonsentinel LN metastases. Conclusions: Increased sentinel LN metastasis lymphangiogenesis is associated with metastatic involvement of nonsentinel axillary LNs. These are the first data sustaining the hypothesis that sentinel LN lymphangiogenesis is involved in further metastatic spread of human breast cancer.

Malignant melanoma of soft parts : further cytogenetic characterization

We report the cytogenetic findings in a case of malignant melanoma of soft parts. Overrepresentation of 1q together with a del(1)(q42), extra copies of chromosomes 7 and 8, and t(12;22)(q13;q13) were found. These findings allow further delineation of the chromosomal pattern found in this uncommon neoplasm.