Peter van Dam

Circulating interleukin-6 predicts survival in patients with metastatic breast cancer.

Interleukin‐6 (IL‐6) is a multifunctional cytokine produced by macrophages, T cells, B cells, endothelial cells and tumour cells. Interleukin‐6 is able to promote tumour growth by upregulating anti‐apoptotic and angiogenic proteins in tumour cells. In murine models it has been demonstrated that antibodies against IL‐6 diminish tumour growth. Several reports have highlighted the prognostic importance of IL‐6 in e.g., prostate and colon cancer. We addressed prospectively the prognostic significance of serum IL‐6 (sIL‐6), measured at diagnosis of metastasis, in 96 unselected and consecutive patients with progressive metastatic breast cancer before the initiation of systemic therapy. The median sIL‐6 value for the breast cancer population was 6.6 ± 2.1 pg/ml. Patients with 2 or more metastatic sites had higher sIL‐6 values compared to those with only 1 metastatic site (respectively 8.15 ± 1.7 pg/ml and 3.06 ± 6.6 pg/ml; p < 0.001). Patients with liver metastasis (8.3 ± 2.4 pg/ml), with pleural effusions (10.65 ± 9.9 pg/ml) and with dominant visceral disease (8.15 ± 3.3 pg/ml) had significantly higher values compared to those without liver metastases (4.5 ± 3.4 pg/ml; p = 0.001), without pleural effusions (5.45 ± 1.5 pg/ml; p = 0.0077) and with dominant bone disease (4.5 ± 1.4 pg/ml; p = 0.007) respectively. No correlation between sIL‐6 and age, menopausal status, performance status, tumour grade, body‐mass index, histology and hormone receptor status was found. Multivariate analysis showed that high levels of serum IL‐6 have independent prognostic value. We conclude that circulating IL‐6 is associated with worse survival in patients with metastatic breast cancer and is correlated with the extent of disease.

Increased Serum Interleukin-8 in Patients with Early and Metastatic Breast Cancer Correlates with Early Dissemination and Survival

Purpose: The prognostic significance of serum interleukin (IL)-8 was evaluated in patients with metastatic breast cancer. The predictive value of serum IL-8 for the presence of occult metastatic tumor cells in bone marrow aspirates was evaluated in patients with operable and metastatic breast cancer. Experimental Design: Serum IL-8 was measured in healthy controls, patients with operable breast cancer, and patients with untreated, progressive metastatic breast cancer. In 69 patients with either operable or advanced breast cancer, occult cytokeratin-positive cells were counted in bone marrow aspirates. Results: Serum IL-8 levels are increased in 67% (52 of 77) of patients with advanced breast cancer. Overall, these levels are significantly higher in patients with breast cancer compared with healthy volunteers (P < 0.001). The IL-8 levels increase significantly in patients with more advanced disease. An elevated serum IL-8 is related to an accelerated clinical course, a higher tumor load, and the presence of liver or lymph node involvement. A multivariate analysis indicates that serum IL-8 is an independent significant factor for postrelapse survival. There was a significant difference between serum IL-8 levels in patients with or without occult cytokeratin-positive bone marrow cells (P < 0.04). Serum IL-8 levels also showed an association with the number of these cells (P < 0.01). Conclusions: Serum IL-8 is increased in patients with breast cancer and has an independent prognostic significance for postrelapse survival. The observations on the relationship between occult cytokeratin-positive bone marrow cells corroborate the concept of IL-8 acting as a contributor to the process of tumor cell dissemination. Similarly, the relationship between serum IL-8 and nodal stage at presentation deserves further study. These results further expand the concept that inflammation and inflammatory cytokines are critical components of tumor progression.

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.

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.

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

Nuclear Factor-κB Signature of Inflammatory Breast Cancer by cDNA Microarray Validated by Quantitative Real-time Reverse Transcription-PCR, Immunohistochemistry, and Nuclear Factor-κB DNA-Binding

Purpose: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer with high metastatic potential. In a previous study, we showed that IBC is a different form of breast cancer compared with non-IBC by cDNA microarray analysis. A list of 756 genes with significant expression differences between IBC and non-IBC was identified. In-depth functional analysis revealed the presence of a high number of nuclear factor-κB (NF-κB) target genes with elevated expression in IBC versus non-IBC. This led to the hypothesis that NF-κB contributes to the phenotype of IBC. The aim of the present study was to further investigate the role of NF-κB in IBC. Experimental Design: Immunohistochemistry and NF-κB DNA-binding experiments were done for all NF-κB subunits (RelA, RelB, cRel, NFkB1, and NFkB2) using IBC and non-IBC specimens. Transcriptionally active NF-κB dimers were identified by means of coexpression analysis. In addition, quantitative real-time reverse transcription-PCR for eight NF-κB target genes, selected upon a significant, 3-fold gene expression difference between IBC and non-IBC by cDNA microarray analysis, was done. Results: We found a significant overexpression for all of eight selected NF-κB target genes in IBC compared with non-IBC by quantitative real-time reverse transcription-PCR. In addition, we found a statistically elevated number of immunostained nuclei in IBC compared with non-IBC for RelB (P = 0.038) and NFkB1 (P < 0.001). Immunohistochemical data were further validated by NF-κB DNA-binding experiments. Significant correlations between immunohistochemical data and NF-κB DNA binding for RelA, RelB, NFkB1, and NFkB2 were found. Transcriptionally active NF-κB dimers, composed of specific combinations of NF-κB family members, were found in 19 of 44 IBC specimens compared with 2 of 45 non-IBC specimens (P < 0.001). In addition, we found evidence for an estrogen receptor (ER)–mediated inhibition of the NF-κB signaling pathway. NF-κB target genes were significantly elevated in ER− versus ER+ breast tumors. Also, the amount of immunostained nuclei for RelB (P = 0.025) and NFkB1 (P = 0.031) was higher in ER− breast tumors versus ER+ breast tumors. Conclusions: The NF-κB transcription factor pathway probably contributes to the phenotype of IBC and possibly offers new options for treatment of patients diagnosed with this aggressive form of breast cancer.

Distinct molecular signature of inflammatory breast cancer by cDNA microarray analysis.

SummaryInflammatory breast cancer (IBC) is a clinically distinct and aggressive form of locally advanced breast cancer with largely unknown genetic determinants. Overexpression of the RhoC GTPase and of HER2, and decreased ER-expression are involved in IBC. Multimodality treatment has increased survival but prognosis is still poor. Novel molecular targets for improved neoadjuvant treatment are necessary. Using cDNA microarrays, we performed genome-wide expression profiling of pre-treatment tumour samples of 16 patients with IBC and 18 patients with non-stage-matched non-IBC. Rigid clinical diagnostic criteria according to the TNM classification of␣the American Joint Committee on Cancer were adopted. Unsupervised hierarchical clustering accurately distinguished IBC and non-IBC samples. A set of 50 discriminator genes was identified in a learning group of tumour samples and was successful in diagnosing IBC in a validation group of samples (accuracy of 88%). Exclusion of ER-related or HER2-related genes did not alter this discriminatory accuracy, indicating that the expression of other genes in addition to ER and HER2 characterize the IBC phenotype. The molecular signature of IBC revealed the overexpression of a large number of NF-κB target genes, explaining at least part of the aggressive nature of IBC. Successful validation of some of the overexpressed genes by immunohistochemistry or real-time quantitative PCR demonstrated the robustness of the cDNA microarray experiments. The results of our study provide potential targets for the treatment of patients with IBC.

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.

Array-based DNA methylation profiling for breast cancer subtype discrimination.

Background Abnormal DNA methylation is well established for breast cancer and contributes to its progression by silencing tumor suppressor genes. DNA methylation profiling platforms might provide an alternative approach to expression microarrays for accurate breast tumor subtyping. We sought to determine whether the distinction of the inflammatory breast cancer (IBC) phenotype from the non-IBC phenotype by transcriptomics could be sustained by methylomics. Methodology/Principal Findings We performed methylation profiling on a cohort of IBC (N = 19) and non-IBC (N = 43) samples using the Illumina Infinium Methylation Assay. These results were correlated with gene expression profiles. Methylation values allowed separation of breast tumor samples into high and low methylation groups. This separation was significantly related to DNMT3B mRNA levels. The high methylation group was enriched for breast tumor samples from patients with distant metastasis and poor prognosis, as predicted by the 70-gene prognostic signature. Furthermore, this tumor group tended to be enriched for IBC samples (54% vs. 24%) and samples with a high genomic grade index (67% vs. 38%). A set of 16 CpG loci (14 genes) correctly classified 97% of samples into the low or high methylation group. Differentially methylated genes appeared to be mainly related to focal adhesion, cytokine-cytokine receptor interactions, Wnt signaling pathway, chemokine signaling pathways and metabolic processes. Comparison of IBC with non-IBC led to the identification of only four differentially methylated genes (TJP3, MOGAT2, NTSR2 and AGT). A significant correlation between methylation values and gene expression was shown for 4,981 of 6,605 (75%) genes. Conclusions/Significance A subset of clinical samples of breast cancer was characterized by high methylation levels, which coincided with increased DNMT3B expression. Furthermore, an association was observed with molecular signatures indicative of poor patient prognosis. The results of the current study also suggest that aberrant DNA methylation is not the main force driving the molecular biology of IBC.

Influence of investigator experience and microscopic field size on microvessel density in node-negative breast carcinoma.

In this study on the determination of intratumoralmicrovessel density (MVD) in breast cancer, we haveinvestigated the influence of the observer experience andthe microscopic field size. We have used thesample set reported on earlier in the JNatl Cancer Inst 87: 1797–1798, 1995. This case-controlstudy has shown a positive association of highMVD and unfavorable outcome when comparing node-negative pT1–2breast carcinoma (NNBC) patients with a disease-free periodof over ten years with those with anearly distant relapse.Tumor sections of both outcome groups (favorable: n= 19; unfavorable: n = 19) were immunostainedfor factor VIII related-antigen (FVIII r-Ag). Microvessels werecounted in the areas of most intense vascularization(‘hot spots’), both at magnification × 200 (fieldsize of 0.61 square mm) and × 400(field size of 0.15 square mm), by oneinexperienced and three experienced observers. Microphotographs of individualvascular hot spots were analyzed using overlays resemblingthe two field sizes.The main results obtained are: i) a confirmationof the prognostic value of microvessel density inthe case-control sample set (n = 38) wasestablished by all experienced but not by theunexperienced investigator; ii) both at × 200 and× 400 magnification, angiogenesis quantification in vascular hotspots contained prognostic information.The results of this study indicate that theselection of vascular hot spots in tumor sectionsimmunostained for an antigen expressed on endothelial cellsis more prone to inter-observer variability and moredependent on training than the counting of themicrovessels within predefined hot spots itself. The microscopicmagnification and resulting field size do not influencethe prognostic significance of MVD in NNBC. Thisinformation validates the development of more objective methodsof measuring the amount of angiogenesis within malignanttissue. This will allow more accurate implementation ofthe angiogenesis parameter in multiparametric and prospective prognosticfactor studies in NNBC.