New data are consistent with a model of direct haematogenous
New data are consistent with a model of direct haematogenous
About 50% of patients with breast cancer have no involvement of axillary lymph nodes at diagnosis and can be considered cured after primary locoregional treatment. However, about 20-30% will experience distant relapse. The group of patients at risk is not well characterised: recurrence is probably due to the establishment of micrometastases before treatment. Given the early steps of metastasis in which tumour cells interact with endothelial cells of blood vessels, and, given the independent prognostic value in breast cancer of both the quantification of tumour vascularisation and the detection of micrometastases in the bone marrow, the aim of this study was to determine the relationship between vascularisation, measured by Chalkley morphometry, and the bone marrow content of cytokeratin-19 (CK-19) mRNA, quantified by real-time reverse transcriptase polymerase chain reaction, in a series of 68 patients with localised untreated breast cancer. The blood concentration of factors involved in angiogenesis (interleukin-6 and vascular endothelial growth factor) and of factors involved in coagulation (D-dimer, fibrinogen, platelets) was also measured. When bone marrow CK-19 relative gene expression (RGE) was categorised according to the cut-off value of 0.77 (95th centile of control patients), 53% of the patients had an elevated CK-19 RGE. Patients with bone marrow micrometastases, on the basis of an elevated CK-19 RGE, had a mean Chalkley count of 7.5 ± 1.7 (median 7, standard error [SE] 0.30) compared with a mean Chalkley count of 6.5 ± 1.7 in other patients (median 6, SE 0.3) (Mann-Whitney U -test; P = 0.04). Multiple regression analysis revealed that Chalkley count, not lymph node status, independently predicted CK-19 RGE status ( P = 0.04; odds ratio 1.38; 95% confidence interval 1.009-1.882). Blood parameters reflecting angiogenesis and coagulation were positively correlated with Chalkley count and/or CK-19 RGE. Our data are in support of an association between elevated relative microvessel area of the primary tumour and the presence of bone marrow micrometastases in breast cancer patients with operable disease, and corroborate the paracrine and endocrine role of interleukin-6 and the involvement of coagulation in breast cancer growth and metastasis.
The development of distant metastases is the primary cause of death in breast cancer patients. The involvement of the axillary lymph nodes, tumour size, histopathological grade and hormone receptor status determine prognosis and treatment options at initial diagnosis. Nevertheless, these parameters do not accurately predict which patients will relapse after primary treatment, and they give limited information about the effectiveness of adjuvant treatment.
About 50% of patients have no involvement of the axillary lymph nodes at diagnosis and can therefore be considered cured after primary locoregional treatment. However, about 20-30% will experience distant relapse within 5-10 years, suggesting outgrowth of disseminated tumour cells present at diagnosis and undetectable by the current diagnostics. This prompted the refinement of methods able to detect subclinical tumour deposits in various body compartments.
Tumour cells residing in bone marrow are considered to mirror the efficacy of the metastatic process throughout the body. Several prospectively designed clinical trials have confirmed the independent prognostic significance of the lodging of tumour cells in the bone marrow, suggesting that this minimal disease is indeed the progenitor of manifest metastasis. It is not clear whether the tumour cells that are part of subclinical metastases have arisen early during progression of the primary tumour or whether they are late and rare metastatic variants as a result of the cumulative acquisition of malignant phenotypic traits such as self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless replicative potential, genomic instability, tissue invasion and sustained angiogenesis.
Bone marrow micrometastasis can be detected by immunocytochemical analyses with antibodies directed at epithelial markers. Polymerase chain reaction (PCR)-based techniques that amplify epithelial mRNA are more sensitive but need the introduction of cut-off values for positivity to correct for the inevitable loss of specificity.
The concept of dependence on vascularisation of growth, invasion and metastasis of malignant tumours has been challenged by the description of angiogenesis-independent mechanisms. Nevertheless, the growth of most primary tumours needs angiogenesis. There is accumulating evidence that angiogenesis is intrinsically linked with the process of haemostasis. Both angiogenesis and haemostasis are tightly regulated in physiological circumstances, for example during wound healing, but are deregulated when involved in tumour growth, invasion and metastasis. We have previously demonstrated the prognostic importance of the angiogenic cytokine interleukin (IL)-6, and the fibrin degradation product D-dimer, in patients with metastatic breast cancer.
A reproducible method of quantifying vascularisation, by assessing the relative microvessel area, is Chalkley point overlap morphometry. Significant associations between the Chalkley count and axillary lymph node metastasis, increasing tumour size, high grade and histological subtype have been reported in patients with breast cancer. Moreover, an independent prognostic value has been demonstrated in patients with breast cancer by a meta-analysis of 87 published studies. Chalkley counting is done in selected areas of high microvessel density, so-called 'hot spots'. The hypothetical rationale for counting in these highly vascular areas is that they predict the presence of more angiogenic subclones of the tumour. These subclones might therefore have a higher metastatic efficiency.
The primary aim of this study was to assess the association of tumour vascularity, quantified by a well-standardised morphometrical method, and the presence of bone marrow micrometastases, detected by a sensitive and quantitative real-time reverse transcriptase PCR (RT-PCR) technique, in patients with operable breast cancer before treatment, to evaluate the relative microvessel area as a surrogate marker of bone marrow micrometastasis, as suggested by Fox and colleagues.
About 50% of patients with breast cancer have no involvement of axillary lymph nodes at diagnosis and can be considered cured after primary locoregional treatment. However, about 20-30% will experience distant relapse. The group of patients at risk is not well characterised: recurrence is probably due to the establishment of micrometastases before treatment. Given the early steps of metastasis in which tumour cells interact with endothelial cells of blood vessels, and, given the independent prognostic value in breast cancer of both the quantification of tumour vascularisation and the detection of micrometastases in the bone marrow, the aim of this study was to determine the relationship between vascularisation, measured by Chalkley morphometry, and the bone marrow content of cytokeratin-19 (CK-19) mRNA, quantified by real-time reverse transcriptase polymerase chain reaction, in a series of 68 patients with localised untreated breast cancer. The blood concentration of factors involved in angiogenesis (interleukin-6 and vascular endothelial growth factor) and of factors involved in coagulation (D-dimer, fibrinogen, platelets) was also measured. When bone marrow CK-19 relative gene expression (RGE) was categorised according to the cut-off value of 0.77 (95th centile of control patients), 53% of the patients had an elevated CK-19 RGE. Patients with bone marrow micrometastases, on the basis of an elevated CK-19 RGE, had a mean Chalkley count of 7.5 ± 1.7 (median 7, standard error [SE] 0.30) compared with a mean Chalkley count of 6.5 ± 1.7 in other patients (median 6, SE 0.3) (Mann-Whitney U -test; P = 0.04). Multiple regression analysis revealed that Chalkley count, not lymph node status, independently predicted CK-19 RGE status ( P = 0.04; odds ratio 1.38; 95% confidence interval 1.009-1.882). Blood parameters reflecting angiogenesis and coagulation were positively correlated with Chalkley count and/or CK-19 RGE. Our data are in support of an association between elevated relative microvessel area of the primary tumour and the presence of bone marrow micrometastases in breast cancer patients with operable disease, and corroborate the paracrine and endocrine role of interleukin-6 and the involvement of coagulation in breast cancer growth and metastasis.
The development of distant metastases is the primary cause of death in breast cancer patients. The involvement of the axillary lymph nodes, tumour size, histopathological grade and hormone receptor status determine prognosis and treatment options at initial diagnosis. Nevertheless, these parameters do not accurately predict which patients will relapse after primary treatment, and they give limited information about the effectiveness of adjuvant treatment.
About 50% of patients have no involvement of the axillary lymph nodes at diagnosis and can therefore be considered cured after primary locoregional treatment. However, about 20-30% will experience distant relapse within 5-10 years, suggesting outgrowth of disseminated tumour cells present at diagnosis and undetectable by the current diagnostics. This prompted the refinement of methods able to detect subclinical tumour deposits in various body compartments.
Tumour cells residing in bone marrow are considered to mirror the efficacy of the metastatic process throughout the body. Several prospectively designed clinical trials have confirmed the independent prognostic significance of the lodging of tumour cells in the bone marrow, suggesting that this minimal disease is indeed the progenitor of manifest metastasis. It is not clear whether the tumour cells that are part of subclinical metastases have arisen early during progression of the primary tumour or whether they are late and rare metastatic variants as a result of the cumulative acquisition of malignant phenotypic traits such as self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless replicative potential, genomic instability, tissue invasion and sustained angiogenesis.
Bone marrow micrometastasis can be detected by immunocytochemical analyses with antibodies directed at epithelial markers. Polymerase chain reaction (PCR)-based techniques that amplify epithelial mRNA are more sensitive but need the introduction of cut-off values for positivity to correct for the inevitable loss of specificity.
The concept of dependence on vascularisation of growth, invasion and metastasis of malignant tumours has been challenged by the description of angiogenesis-independent mechanisms. Nevertheless, the growth of most primary tumours needs angiogenesis. There is accumulating evidence that angiogenesis is intrinsically linked with the process of haemostasis. Both angiogenesis and haemostasis are tightly regulated in physiological circumstances, for example during wound healing, but are deregulated when involved in tumour growth, invasion and metastasis. We have previously demonstrated the prognostic importance of the angiogenic cytokine interleukin (IL)-6, and the fibrin degradation product D-dimer, in patients with metastatic breast cancer.
A reproducible method of quantifying vascularisation, by assessing the relative microvessel area, is Chalkley point overlap morphometry. Significant associations between the Chalkley count and axillary lymph node metastasis, increasing tumour size, high grade and histological subtype have been reported in patients with breast cancer. Moreover, an independent prognostic value has been demonstrated in patients with breast cancer by a meta-analysis of 87 published studies. Chalkley counting is done in selected areas of high microvessel density, so-called 'hot spots'. The hypothetical rationale for counting in these highly vascular areas is that they predict the presence of more angiogenic subclones of the tumour. These subclones might therefore have a higher metastatic efficiency.
The primary aim of this study was to assess the association of tumour vascularity, quantified by a well-standardised morphometrical method, and the presence of bone marrow micrometastases, detected by a sensitive and quantitative real-time reverse transcriptase PCR (RT-PCR) technique, in patients with operable breast cancer before treatment, to evaluate the relative microvessel area as a surrogate marker of bone marrow micrometastasis, as suggested by Fox and colleagues.
