Occult micrometastasis: enrichment, identification and characterization of single disseminated tumour cells

The decision as to whether systemic adjuvant therapy should be applied in breast cancer patients for secondary prevention of metastatic relapse is based solely on the statistical prognosis. For this reason, the direct identification of minimal residual cancer in distant organs (e.g. bone marrow) is of particular importance. In breast cancer 25-43% of the patients exhibit micrometastatic disease in bone marrow, following resection of their primary tumours. Successful enrichment, reliable identification and molecular profiling of disseminated tumour cells at the single cell level are still key issues in ongoing and future studies. In addition, first attempts have been reported to evaluate the biology of disseminated tumour cells using in vitro and in vivo models. Taken together, the advancing characterization of disseminated tumour cells opens the avenue for the development of new therapeutic approaches aimed at preventing metastatic relapse.     

Micrometastatic tumour cells in bone marrow of patients with gastric cancer: Methodological aspects of detection and prognostic significance

Monoclonal antibodies (Mab) are potent probes to identify individual tumour cells or small tumour cell clusters in bone marrow. In the present study, various antibodies directed against either cell surface or intracytoplasmic antigens of epithelial cells were assessed for their ability to detect such cells in bone marrow of patients with breast, colorectal and gastric cancer. According to the presented data, monoclonal antibodies against intracellular cytokeratin (CK) components are superior in terms of specificity and sensitivity to antibodies reacting with epitopes of the cell membrane. Using a monoclonal antibody against the cytokeratin polypeptide 18 in connection with the alkaline phosphatase anti-alkaline phosphatase detection system (APAAP), we could detect tumour cells in bone marrow of 34 out of 97 patients with gastric cancer examined at the time of primary surgery. The incidence of positive findings was correlated to established risk factors, such as histological classification and locoregional lymph node involvement. Clincal follow-up studies on 38 patients demonstrated a significantly increased relapse rate in patients presenting with CK-positive cells in their bone marrow at the time of primary surgery. Thus the described technique may help to identify patients with gastric cancer carrying a high risk of early relapse.     

Detection of disseminated tumour cells in bone marrow of patients with isolated liver metastases from colorectal cancer

BACKGROUND AND OBJECTIVES: In colorectal cancer (CRC) patients, tumour recurrence is common following potentially curative surgery for liver metastases. This may be caused by occult tumour cells present at the time of surgery. Dissemination of micrometastatic cells may occur early in patients with solid cancer, and micrometastases may signify a poor prognosis. The aim of the present study was to evaluate the frequency of micrometastatic cells in the bone marrow of patients with potentially resectable liver metastases. METHODS: Twenty millilitres of bone marrow was aspirated from both anterior iliac crests from 48 patients. Mononuclear cells were isolated and incubated with superparamagnetic Dynabeads coated with an anti-epithelial monoclonal antibody (MOC31). Magnetically selected cells were identified by light microscopy as cells with bead rosettes (>5 beads/cell). RESULTS: Micrometastatic tumour cells were identified in four of 48 (8%) patients who all had their liver metastases surgically removed. Two of the four died after 17 and 18 months, respectively, whereas two are alive after 10 and 12 months. None of the 19 inoperable patients had micrometastases. CONCLUSIONS: The frequency of bone marrow micrometastases in patients with clinically isolated liver metastases from CRC was low. This is biologically interesting, but bone marrow status should not affect current treatment protocols.     

Tumour cell detection in the bone marrow of breast cancer patients at primary therapy: results of a 3-year median follow-up.

We examined bone marrow aspirates from 100 metastasis-free primary breast cancer patients. In 38/100 patients (38%), tumour cells were detected in the marrow using an immunocytochemical technique with a cocktail of two monoclonal antibodies: anti-EMA and anti-cytokeratin. Median follow-up was 34 months: 15/38 (39%) tumour cell-positive patients have since relapsed, but only 9/62 (15%) tumour cell-negative patients. The median interval between tumour cell detection and relapse was 11.4 months. No statistically significant correlation existed between tumour cell presence and ''established'' prognostic factors. However, relapse-free survival was significantly shorter in tumour cell-positive patients. Multivariate analysis showed tumour cell presence as a strong, significant prognostic factor for relapse-free as well as overall survival. We conclude that screening for tumour cells in bone marrow of primary breast cancer patients identifies high-risk patients for early relapse. In particular, patients with node-negative tumours who have tumour cells in their bone marrow may require subsequent systemic therapy.     

Bone marrow leukemic progenitor cell content in pediatric T-lineage acute lymphoblastic leukemia patients with an isolated extramedullary first relapse.

Isolated extramedullary relapse in childhood acute lymphoblastic leukemia (ALL) is associated frequently with the T-lineage immunophenotype and may be accompanied by occult bone marrow disease. We employed highly sensitive multiparameter flow cytometry and blast colony assays to quantify the leukemic progenitor cell (LPC) burden in the pretreatment bone marrows of 15 pediatric T-lineage ALL patients with an isolated extramedullary first relapse. Sites of extramedullary relapse were CNS (11 patients), testes (3 patients), and both CNS and testes (1 patient). Bone marrow LPC were detectable in 8 patients (53%) and undetectable in 7 patients (47%) at day 0 of post-relapse induction therapy, with LPC counts ranging from 0/10(6) mononuclear cells (MNC) to 518/10(6) MNC (mean +/- SEM, 50+/-34/10(6) MNC). Five of 9 patients with an early relapse (< 18 months after achieving a first complete remission [CR1]) and 3 of 6 patients with a late relapse (> or = 18 months from CR1) had detectable bone marrow LPC at day 0. Five of 8 patients with NCI-defined poor risk ALL and 3 of 7 patients with NCI-defined standard risk ALL had detectable LPC at day 0. Following post-relapse induction chemotherapy. LPC counts were detectable in bone marrows of 4 of 6 evaluated patients. Thus, approximately half of the extramedullary relapse T-lineage ALL patients studied had substantial occult involvement of the bone marrow. These findings may partly explain the previously observed poor prognosis of T-lineage patients following a CNS relapse.     

Clinical significance of disseminated tumour cells in non-small cell lung cancer

Background:

Early-stage non-small cell lung cancer (NSCLC) patients have a high risk of disease relapse despite curatively intended surgical resection, and the detection of tumour cells in the bone marrow could be one method of determining the presence of the disseminated disease in its early stages.

Methods:

Bone marrow aspirates were collected from 296 patients at the time of surgery, and the presence of disseminated tumour cells was determined with the help of immunomagnetic selection (IMS) using the MOC31-antibody recognising EpCAM and with the help of standard immunocytochemistry (ICC) using the anti-cytokeratin (CK) antibodies AE1/AE3.

Results:

Disseminated tumour cells were found in 152 of 252 (59%) bone marrow samples using IMS and in 25 of 234 (11%) samples using ICC. No association between the two detection methods was observed. The presence of EpCAM⁺ cells was not associated with any clinicopathological parameters, whereas a higher frequency of CK⁺ cells was found in patients with an advanced pT status. Disseminated tumour cells, as detected using IMS, had no prognostic impact. Patients with CK⁺ cells in the bone marrow had a reduced relapse-free survival, but the difference was not statistically significant.

Conclusion:

Our findings do not support the further development of DTC detection for clinical use in early-stage NSCLC. Future studies should include the molecular characterisation of DTCs, along with an attempt to identify subpopulations of cells with biological and clinical significance.     

Clinical relevance of circulating tumour cells in the bone marrow of patients with SCCHN

BACKGROUND: Clinical outcome of patients with head and neck squamous cell carcinoma (SCCHN) depends on several risk factors like the presence of locoregional lymph node or distant metastases, stage, localisation and histologic differentiation of the tumour. Circulating tumour cells in the bone marrow indicate a poor prognosis for patients with various kinds of malignoma. The present study examines the clinical relevance of occult tumour cells in patients suffering from SCCHN. PATIENTS AND METHODS: Bone marrow aspirates of 176 patients suffering from SCCHN were obtained prior to surgery and stained for the presence of disseminated tumour cells. Antibodies for cytokeratin 19 were used for immunohistochemical detection with APAAP on cytospin slides. Within a clinical follow-up protocol over a period of 60 months, the prognostic relevance of several clinicopathological parameters and occult tumour cells was evaluated. RESULTS: Single CK19-expressing tumour cells could be detected in the bone marrow of 30.7% of the patients. There is a significant correlation between occult tumour cells in the bone marrow and relapse. Uni- and multivariate analysis of all clinical data showed the metastases in the locoregional lymph system and detection of disseminated tumour cells in the bone marrow to be statistically highly significant for clinical prognosis. Conclusion: The detection of minimal residual disease underlines the understanding of SCCHN as a systemic disease. Further examination of such cells will lead to a better understanding of the tumour biology, as well as to improvement of diagnostic and therapeutic strategies.     

Antigenic profiles of disseminated breast tumour cells and microenvironment in bone marrow.

AIMS: Thirty per cent of breast cancer patients with axillary lymph node negative at primary surgery will relapse within 10 years. This may be caused by disseminated tumour cells from the primary tumour. This study report the phenotypic profiles of disseminated tumour cells and microenvironmental characteristics in bone marrow of breast cancer. METHODS: We detected the biologic markers on the disseminated tumour cells with immunocytochemical staining, analysed the immunological changes through flow-cytometry, and investigated the u- PA activity in the plasma of bone marrow. RESULTS: With the immunocytochemical staining of EMA and CK19, we detected micrometastasis in thirty out of 72 (41.67%) breast cancer patients. Compared with the primary tumours, disseminated tumour cells expressed low protein cyclin D1, P53, Ki-67, EGFR, and high protein P21. The percentage of memory CD4+ T cells was significantly higher in the micrometastasis-positive group than in the micrometastasis-negative group. Tumour size and axillary lymph node status were found to be significantly correlated with the u- PA activity level. CONCLUSIONS: Immunophenotypic profiles of disseminated tumour cells could be measured by immunocytochemical staining and microenvironment can be analysed by flow cytometry.     

Adjuvant bisphosphonates in breast cancer: Are we witnessing the emergence of a new therapeutic strategy?

Great strides have been made over the last 20 years in the treatment of breast cancer and, despite an increasing incidence, the number of deaths has fallen sharply since the late 1980s. The widespread use of new adjuvant therapies including trastuzumab, taxanes and aromatase inhibitors should decrease this even further. However, for women, metastatic breast cancer still remains the number one cause of cancer death in Europe, and the detection and treatment of micrometastatic disease represent the most important challenge in breast cancer management.Bone is the most frequent site of distant relapse, accounting for 30–40% of all first recurrence. In addition to the well-recognised release of bone cell activating factors from the tumour, a wealth of pre-clinical data indicates that the release of bone-derived growth factors and cytokines into the microenvironment can both attract cancer cells to the bone surface and facilitate their growth and proliferation. Bisphosphonates are potent inhibitors of bone osteolysis and may interrupt this so-called ‘viscous cycle’, thereby impeding both the development of bone metastases and the survival of dormant cells in the marrow microenvironment for the subsequent dissemination to extra-osseus sites. Additionally, the potent amino-bisphosphonates may also have direct effects on tumour cells, especially when administered in combination with chemotherapy.Clinical trial results with the early oral bisphosphonate and clodronate were judged to be inconclusive but the recent data with zoledronic acid suggest that bone targeted treatments may indeed modify the course of the disease. The results of ongoing large metastasis prevention trials are however, required before routine use of adjuvant bisphosphonates can be recommended.     

Pourquoi certains cancers métastasent-ils préférentiellement à l’os ?

Bone metastases are common complications of several cancers, among which breast and prostate cancer are the most osteotropic. Different factors produced by the bone microenvironment draw cancer cells to invade the bone marrow where they seed in specific regions termed the premetastatic niche and the osteoblastic niche. Cancer cells then acquire bone-like properties to adapt and thrive in the bone microenvironment (osteomimicry). The inhibition of these mechanisms may offer new therapeutic perspectives, preventing bone metastasis formation inpatients at high risk of relapse in bone.     

Surgical cytoreduction in recurrent ovarian carcinoma in patients with complete response to paclitaxel-platinum.

AIM: The objective was to analyse the impact of secondary cytoreductive surgery in patients with recurrent ovarian carcinoma. METHODS: Retrospective review of 572 consecutive patients with primary ovarian carcinoma. Thirty-eight patients with intraabdominal/pelvic recurrence consisted the study group. Clinical variables affecting tumour resectability and survival were evaluated. RESULTS: Complete tumour resection was obtained in 42% of patients. A solitary tumour recurrence was independently associated with complete tumour resection (p=0.009). Median survival for patients with complete and incomplete tumour resection was 51.8 and 19.9 months. The parameter, residual tumour, was found independently correlated with survival after the relapse surgical procedure (p=0.02). However, including also the parameter, number of relapse tumour sites, in the multivariate analysis, the parameter, residual tumour, was no longer significantly associated with survival. CONCLUSIONS: Complete tumour resection following secondary cytoreductive surgery is associated with improved survival in selected groups of patients with recurrent ovarian cancer. However, other clinical factors than surgical cytoreduction are of considerable significance in determining the outcome of the salvage treatment.     

Removal of tumour cells from bone marrow: an evaluation of the available techniques

Autologous bone marrow transplantation has offered a way of increasing the dose of drugs and radiotherapy which can be used to treat patients with malignant disease in an attempt to eradicate tumour. Bone marrow is taken prior to treatment and returned to the patient to 'rescue' haemopoietic function after ablative therapy is completed. Bone marrow removed for autograft may be contaminated with tumour cells at the time of harvest, and it is undesirable to return these to patients even though there are little data available concerning the number of tumour cells necessary to reseed various malignancies. This review considers the various methods available for removing tumour cells from bone marrow destined for autologous transplantation, and evaluates their advantages and disadvantages.     

Analysis of and prognostic information from disseminated tumour cells in bone marrow in primary breast cancer: a prospective observational study

ABSTRACT: BACKGROUND: Disseminated tumour cells (DTCs) in the bone marrow of patients with breast cancer have been identified as an independent predictor of poor prognosis in patients with non-metastatic disease. This prospective study aimed to evaluate the presence and prognostic value of DTCs in the bone marrow of female patients with primary breast cancer. METHODS: Between 1999 and 2003, bone marrow aspirates were obtained from patients at the time of surgery for primary invasive breast cancer. DTCs in bone marrow were identified using monoclonal antibodies against cytokeratins for detection of epithelial cells. The detection of DTCs was related to clinical follow-up with distant disease-free survival (DDFS) and breast cancer-specific survival as endpoints. Bone marrow aspirates from adult healthy bone marrow donors were analysed separately. RESULTS: DTCs were analysed in 401 patients, and cytokeratin-positive cells were found in 152 of these (38%). An immunofluorescence (IF) staining procedure was used in 327 patients, and immunocytochemistry (IC) was performed in 74 patients. The IF-based method resulted in 40% DTC-positive cases, whereas 30% were positive using IC (p = 0.11). The presence of DTCs in bone marrow was not significantly related to patient or tumour characteristics. The presence of DTCs was not a prognostic factor for DDFS (IF: hazards ratio [HR], 2.2; 95% confidence interval [CI], 0.63--2.2; p = 0.60; IC: HR, 0.84; 95% CI, 0.09--8.1; p = 0.88). Significant prognostic factors were lymph node metastases, oestrogen receptor positivity, Nottingham histological grade, and tumour size using Cox univariate analysis. The analyses were positive for epithelial cells in bone marrow from adult healthy donors in 19 (25%) samples. CONCLUSIONS: The detection of DTCs in bone marrow in primary breast cancer was previously shown to be a predictor of poor prognosis. We were not able to confirm these results in a prospective cohort including unselected patients before the standard procedure was established. Future studies with a standardised patient protocol and improved technique for isolating and detecting DTCs may reveal the clinical applications of DTC detection in patients with micrometastases in the bone marrow.     

Prognostic significance of an increased number of micrometastatic tumor cells in the bone marrow of patients with first recurrence of breast carcinoma

BACKGROUND: Using cytokeratin (CK) as a histogenetic marker of epithelial tumor cells in the bone marrow of patients with primary breast carcinoma, a subgroup of patients with decreased survival can be identified. This study was designed to evaluate the frequency and prognostic relevance of such cells in patients with recurrent breast carcinoma. METHODS: Bone marrow aspirates from 65 patients were analyzed immunocytochemically for the presence of CK positive cells. A quantitative immunoassay with monoclonal anti-CK antibody A45-B/B3 was used and 2 x 10(6) bone marrow cells per patient were evaluated. For prognostic evaluation the authors calculated a cutoff value of micrometastatic tumor cells by analogy to classification and regression tree (CART) analysis. Patients were monitored prospectively for a median of 37 months (range, 11-63 months). RESULTS: Bone marrow micrometastases were present in 5 of 32 patients (16%) with locoregional recurrence and in 24 of 33 patients (73%) with distant recurrence. The bone marrow status yielded no prognostic indication for patients with locoregional recurrence. In contrast, a cutoff value of 2.5 tumor cells per 1 million bone marrow cells analyzed (2.5 x 10(-6) tumor cells) correlated with a significantly different prognosis for women with distant disease. Patients with metastatic disease and a micrometastatic tumor load of > 2.5 x 10(-6) tumor cells survived for a mean of 6 months (95% confidence interval [95% CI], 2.0-9.1) compared with 17 months (95% CI, 11.6-22.0) for patients with < or = 2.5 x 10(-6) tumor cells (P < 0.0001). Multivariate analysis, allowing for hormone receptor status, disease free interval prior to recurrence, manifestation site of metastases, age, and micrometastases in bone marrow, revealed that bone marrow involvement was an independent risk factor, with a hazard ratio of 7.4 (95% CI, 1.6-13.3) for disease-related death. CONCLUSIONS: An increased number of micrometastases identified in the bone marrow of patients with metastatic breast carcinoma represents an independent prognostic factor that may influence future therapeutic strategies for patients with metastatic breast carcinoma.     

Tumor cell dissemination in colon cancer does not predict extrahepatic recurrence in patients undergoing surgery for hepatic metastases

Patients undergoing resection of hepatic metastases of colorectal cancer have a high risk of extrahepatic recurrence, most likely caused by early tumor cell dissemination or the manipulation of liver tumors during surgical resection. Using immunocytochemistry, we studied 47 patients for cytokeratin (CK)-positive (+) cells in: a) bone marrow (BM) samples to determine whether tumor cell dissemination had already occurred before surgery; and b) blood samples directly taken from the hepatic vein before and during surgery of liver metastases. In addition, normal and malignant liver tissues were evaluated for markers known to be involved in tumor progression and metastasis [urokinase plasminogen activator (uPA), Her-2/neu, epidermal growth factor receptor (EGF-R)] using sandwich enzyme immunoassays. CK+ cells were detected in the BM of 26/47 patients (55%), in blood samples of 14/47 patients (30%) before surgery and 11/47 patients (23%) during surgery with a median detection rate of 1 (range, 1-14) CK+ cell per 4x10(6) MNC. No CK+ cells were found in 15/47 patients (32%) in any sample studied. Tumor tissue was obtained from 32/47 patients and normal liver tissue from 24/32 patients. While no differences were found for EGF-R and Her-2/neu, a 9-fold higher expression of uPA could be demonstrated in tumor tissue of 20/32 patients (63%) compared to normal liver tissue. When all obtained results were correlated with clinical outcome, neither the detection of CK+ cells nor the expression pattern in the tumor tissue, or the combination of both, was predictive for extrahepatic recurrence or overall survival after a mean observation time of 43 months (range, 26-54 months). Although uPa is overexpressed in liver metastases of colorectal cancer, and dissemination of CK+ cells during surgery of these metastases is a frequent event in colon cancer, these findings do not predict extrahepatic recurrence. Further characterization of single cells, especially those spread during surgery, will help to identify those patients with an increased risk of later relapse.     

Tumour T-lymphocyte subset infiltration and tumour recurrence following curative resection for colorectal cancer.

AIM: The relationship of tumour T-lymphocytic subset infiltration and recurrence in patients undergoing potentially curative resection for colorectal cancer has not been clearly defined. METHODS: Tumour sections from patients who had undergone potentially curative resection for colorectal cancer were stained and counted for CD4+ and CD8+ T-lymphocytes. RESULTS: Twenty-three patients developed recurrence during the follow-up period. Patients were grouped according to whether or not they developed recurrence. The groups were similar in age, sex, site of tumour, Dukes stage and the numbers of patients receiving adjuvant therapy. The total percentage volume of labelled CD4+ T-lymphocytes in the tumour was significantly lower in the patients who recurred (p<0.05). CONCLUSIONS: The results of the present pilot study suggest that a reduction in tumour T-lymphocyte infiltration, in particular CD4+ T-lymphocyte infiltration, is associated with recurrence in patients following potentially curative resection for colorectal cancer.     

Sequential immunochemotherapy and edrecolomab in the adjuvant therapy of breast cancer: reduction of 17-1A-positive disseminated tumour cells.

BACKGROUND: The aim of our study was to evaluate the efficacy of the monoclonal antibody edrecolomab after chemo- and radiotherapy in the elimination of disseminated tumour cells in bone marrow in the adjuvant therapy of breast cancer. PATIENTS AND METHODS: The bone marrow of 25 patients with breast cancer was tested for the presence of disseminated tumour cells using the pancytoceratine antibody and the alkaline phosphatase-anti-alkaline-phosphatase (APAAP) technique. To characterize tumour cells simultaneously, immunofluorescent double labelling of pancytoceratine and epithelial cell adhesion molecule (antibody 17-1A) was performed on tumour cells after magneto bead enrichment. Patients positive for the 17-1A antigen in bone marrow after chemotherapy were treated with edrecolomab (500 mg Panorex) initially, then 100 mg/month over 4 months) and investigated for the presence of micrometastases 6 weeks after the last treatment. RESULTS: Of the 17 patients showing bone marrow micrometastases (BM-MM), 14 tested 17-1A positive before adjuvant chemotherapy. After chemotherapy, nine patients remained positive for the 17-1A antigen and were treated with edrecolomab. The final investigation after immunotherapy showed a complete elimination of the 17-1A-positive BM-MM in seven patients and a significant reduction of these cells in two patients. CONCLUSIONS: Sequential treatment of breast cancer with edrecolomab after adjuvant chemotherapy can reduce disseminated tumour cells in the bone marrow and eliminate 17-1A-positive micrometastases.