Ina Benoy

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.

Platelet number and interleukin-6 correlate with VEGF but not with bFGF serum levels of advanced cancer patients

SummaryWe have compared the platelet number and the serum concentration of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and interleukin-6 (IL-6) in 80 blood samples of 50 patients with advanced cancer. We have also measured the mitogenic effect of patient sera on endothelial cells in vitro in order to estimate the biological activity of serum VEGF. Serum VEGF concentration correlated with platelet number (r = 0.61; P < 10–4). Serum IL-6 levels correlated with platelet count (r = 0.36; P < 10–3), with serum VEGF levels (r = 0.55; P < 10–4) and with the calculated load of VEGF per platelet (r = 0.4; P = 3 × 10–4). Patients with thrombocytosis had a median VEGF serum concentration which was 3.2 times higher (P < 10–4) and a median IL-6 serum level which was 5.8 times higher (P = 0.03) than in other patients. Serum bFGF did not show an association with any of the other parameters. Patient sera with high VEGF and bFGF content stimulated endothelial cell proliferation significantly more than other sera (P = 4 × 10–3). These results support the role of platelets in the storage of biologically active VEGF. Platelets seem to prevent circulating VEGF from inducing the development of new blood vessels except at sites where coagulation takes place. IL-6, besides its thrombopoietic effect, also seems to affect the amount of VEGF stored in the platelets. This is in accordance with the indirect angiogenic action of IL-6 reported previously. The interaction of IL-6 with the angiogenic pathways in cancer might explain the stimulation of tumour growth occasionally observed during IL-6 administration. It also conforms to the worse outcome associated with high IL-6 levels and with thrombocytosis in several tumour types and benign angiogenic diseases.

Real-time RT-PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer

This study assessed the ability of real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n=116/78%) and metastatic (stage M+, n=32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT–PCR detection method. The mean follow-up time was 786 days (+/− 487). Kaplan–Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P=0.0045 (CK-19) and P=0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P=0.551 (CK-19) and P=0.329 (MAM)). Separate analyses of the M0 and M+ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M+ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results.

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer

Background:The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardisation of technology hampers the implementation of CTC measurement in clinical routine practice.Methods:This study was designed to directly compare three techniques for detecting CTCs in blood samples taken from 76 patients with metastatic breast cancer (MBC) and from 20 healthy controls: the CellSearch CTC System, the AdnaTest Breast Cancer Select/Detect and a previously developed real-time qRT-PCR assay for the detection of CK-19 and mammaglobin transcripts.Results:As a result, 36% of patients with MBC were positive by the CellSearch System, 22% by the AdnaTest, 26% using RT–PCR for CK-19 and 54% using RT–PCR for mammaglobin. Samples were significantly more likely to be positive for at least one mRNA marker using RT–PCR than using the CellSearch System (P=0.001) or the AdnaTest (P<0.001).Conclusion:We observed a substantial variation in the detection rates of CTCs in blood from breast cancer patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and mammaglobin, which suggests that this is currently the most sensitive technique for detecting CTCs.

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.

Platelets and vascular endothelial growth factor (VEGF): A morphological and functional study

The growth of primary tumours beyond a critical mass is dependent on angiogenesis. The switch to the angiogenic phenotype involves changes in the local equilibrium of cytokines with either pro- or anti-angiogenic properties. Vascular Endothelial Growth Factor (VEGF) is one of the major positive regulators of tumour angiogenesis. Serum VEGF is, in cancer patients, correlated with worse prognosis. Recent evidence suggests that platelets are the main contributors of serum VEGF. We demonstrate, ultrastructurally and with immunofluorescence techniques, the alpha granule and membranous localisation of VEGF and provide further evidence for the role of platelets, both in healthy individuals as in patients with locally and advanced breast cancer, in the storage of circulating VEGF. We also demonstrate that, linear with tumoural progression, platelets accumulate more VEGF. Enhanced production in bone marrow platelet progenitors as well as endocytosis of circulating VEGF by platelets and/or megakaryocytes could explain the higher VEGF load in platelets from advanced cancer patients. This study provides further evidence for a role of platelets in transporting VEGF.

Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types

The causal relationship between persistent infection with high‐risk HPV and cervical cancer has resulted in the development of HPV DNA detection systems. The widely used MY09/11 consensus PCR targets a 450bp conserved sequence in the HPV L1 gene, and can therefore amplify a broad spectrum of HPV types. However, limitations of these consensus primers are evident, particularly in regard to the variability in detection sensitivity among different HPV types. This study compared MY09/11 PCR with type‐specific PCRs in the detection of oncogenic HPV types. The study population comprised 15, 774 patients. Consensus PCR failed to detect 522 (10.9%) HPV infections indicated by type‐specific PCRs. A significant correlation between failure of consensus PCR and HPV type was found. HPV types 51, 68 and 45 were missed most frequently. The clinical relevance of the HPV infections missed by MY09/11 PCR was reflected in the fraction of cases with cytological abnormalities and in follow‐up, showing 104 (25.4%) CIN2+ cases. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. Diagnostic accuracy of the PCR systems, in terms of sensitivity (MY09/11 PCR: 87.9%; type‐specific PCRs: 98.3%) and specificity (MY09/11 PCR: 38.7%; type‐specific PCRs: 76.14%), and predictive values for histologically confirmed CIN2+, suggest that type‐specific PCRs could be used in a clinical setting as a reliable screening tool.

Blood platelets and serum VEGF in cancer patients.

We read with interest the publication of Banks et al (1998) on the significance of platelets for vascular endothelial growth factor (VEGF) measurements. We disagree, however, with the conclusion that for accurate measurement of circulating VEGF, serum is completely unsuitable. We are also convinced that platelets are not the sole origin of circulating VEGF. We would like to stress the potential clinical usefulness of the determination of serum VEGF levels in cancer patients based on published data and on some recent original observations. In the studies described below, the enzyme-linked immunosorbent assay (ELISA) methodology used by Banks et al (1998) was applied. In 44 untreated patients with disseminated colorectal cancer, elevated serum VEGF, i.e. above 500 pg ml–1, was significantly associated with fast tumour growth, i.e. estimated volume doubling time of less than 6 months (Dirix et al, 1996), independent of the number or site of metastasis, suggesting a predictive value of serum VEGF for the progression of the disease. In 42 treated metastatic cancer patients, including patients with colorectal, breast, ovarian and renal carcinomas, a significantly higher fraction, i.e. 32%, of patients with progressive disease during chemotherapeutic treatment had elevated serum VEGF compared with 15% of patients showing response to treatment (Dirix et al, 1997). In 82 patients with non-Hodgkin’s lymphoma, followed up for at least 5 years, 71% of patients with lower than the median serum VEGF before treatment had a 5-year survival rate compared with only 49% among patients with a higher than the median serum VEGF (P = 0.01) (Salven et al, 1997a). In small-cell-lung cancer (n = 68), a high pretreatment serum VEGF predicts poor response to chemotherapy (P = 0.008) and shorter survival (P = 0.01) (Salven et al, 1998). The results of these and other clinical studies (Salven et al, 1997b; Vermeulen et al, 1997) suggest the potential relevance of measuring serum VEGF concentrations in cancer patients. Because of the association of serum VEGF concentration and platelet count in 27 breast cancer patients reported by Verheul et al (1997) and the increase in VEGF concentration after activation of platelet-rich plasma reported by Banks et al (1998), we have compared the platelet count with serum VEGF and basic fibroblast growth factor (bFGF) levels in 58 patients (142 measurements). We found an association between platelet count and serum VEGF (r = 0.50; 95% confidence interval 0.37–0.61; P < 0.0001) (Figure 1), but not between platelet count and serum bFGF (r = 0.06; P = 0.5). In 12 patients for whom we had four or more blood samples at different times during treatment or follow-up, we analysed the intrapatient association of serum VEGF and platelet count. In 10 out of 12 patients (83%), the blood sample with the highest platelet count also had the highest serum VEGF level, although this was the case for only 5 out of 12 patients (42%) for serum bFGF. Changes in serum VEGF, i.e. increase or decrease, were congruent with changes in platelet count in 82% of 39 changes. For serum bFGF, this was the case for only 59% of all changes.