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.     

Prognostic relevance of urokinase plasminogen activator detection in micrometastatic cells in the bone marrow of patients with primary breast cancer.

Patients with an elevated level of urokinase plasminogen activator (uPA) in breast cancer tissue have an adverse prognosis. This study evaluated the prognostic relevance of uPA detection in disseminated tumour cells in bone marrow. Bone marrow was sampled intraoperatively from both iliac crests in 280 patients with primary breast cancer. Interphase cells were enhanced and stained immunocytologically with two antibodies: 2E11, which detects TAG 12--a tumour-associated glycoprotein typically expressed by almost all breast cancer cells--and the anti-uPA antibody HD-UK9. Thirty-five of the 2E11-positive women (n = 132, 47%) developed metastatic disease (median follow-up time 44 months). Of these, most were uPA positive (n = 23, 65%) and only 12 were uPA negative. Patients with uPA-positive cells in bone marrow (n = 98, 35%) had a significantly shorter metastasis-free interval (36 months) than women who were uPA negative (44.5 months). The worst prognosis was seen in patients positive for both markers (29.5 months), followed by those who were uPA negative and 2E11 positive (37 months). The detection of uPA on disseminated tumour cells characterizes a subgroup of patients with an even worse prognosis, who should undergo more aggressive adjuvant systemic therapy. For the first time, it was possible to evaluate an important qualitative parameter involved in the process of breast cancer metastases.     

Relevance of disseminated tumour cells in blood and bone marrow of patients with solid epithelial tumours in perspective.

Currently-used systems to predict prognosis in patients with solid epithelial tumours after surgical resection of the tumour do not give any guarantees for the individual patient. In this respect the clinical relevance of the presence of disseminated tumour cells in blood and bone marrow has been frequently studied. Because of growing awareness that information on merely the presence of disseminated tumour cells is not sufficient for prognostic and therapeutic purposes, attention for characterization of disseminated tumour cells has increased. Numerous reviews have already been published on the detection and clinical relevance of disseminated tumour cells. Therefore, this paper will mainly focus on the biological significance of these cells and discusses the (in)efficiency of the metastatic process, the genotypic and phenotypic characteristics of disseminated tumour cells, and their structure of appearance. Despite the fact that information gained on the several individual aspects is substantial, it did not render any solid solutions for individual patient management yet. Hence, a combined approach of several aspects of disseminated tumour cells together with characteristics and behaviour of the primary tumour is needed to substantially improve our knowledge on the role of disseminated tumour cells in the complex process of tumour metastasis.     

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 relevance of cathepsin D detection in micrometastatic cells in the bone marrow of patients with primary breast cancer

Patients with an elevated level of cathepsin D in breast cancer tissue have an adverse prognosis. This study evaluated the prognostic relevance of cathepsin D detection in disseminated tumour cells in bone marrow. Bone marrow was sampled intraoperatively from both anterior iliac crests in 290 patients with primary breast cancer. Interphase cells were enhanced and stained immunocytologically with two antibodies: BM2, which detects tumour-associated glycoprotein TAG 12, which is typically expressed by almost all breast cancer cells, and the anti-cathepsin D antibody. 67 of 149 BM2-positive women (45%) developed metastatic disease (median follow-up time: 69 months). Of these, 15 were cathepsin D-positive (22%). Patients with cathepsin D-positive cells in bone marrow (n = 26; 9%) had a significantly shorter metastasis-free interval (38 months) compared with women who were cathepsin D-negative (64.5 months). The worst prognosis was seen in patients positive for both markers (30.5 months), followed by those who were cathepsin D-negative and BM2-positive (48 months). The detection of cathepsin D on disseminated tumour cells characterises a subgroup of patients with a poorer prognosis who should undergo more aggressive adjuvant systemic therapy.     

Clinical significance of proliferative potential of occult metastatic cells in bone marrow of patients with breast cancer

There is increasing statistical evidence that the presence of tumour cells in bone marrow detected by immunocytochemistry represents an important prognostic indicator in breast cancer, but their individual capacity to become clinical metastases is unknown. The aim of this study was to assess the proliferative capacity of these occult metastatic cells in the bone marrow of patients with various stages of breast cancer. We obtained bone marrow aspirates from 60 patients with breast cancer before treatment with chemotherapy: 17 stage II, 12 stage III and 31 stage IV. After bone marrow culture for 6-34 days (median: 17 days) under specific cell culture conditions, viable epithelial cells were detected by cytokeratin staining in 40 patients (66%). Expansion of tumour cells was poorly correlated with tumour cell detection on primary screening (P=0.06). There was a nonsignificant correlation between the number and the presence of expanded tumour cells and the UICC stage of the patients. On primary screening, tumour cell detection was positive in 56% of patients and was correlated with clinical UICC stage (P=0.01). However, with a median follow-up of 23 months, expansion of tumour cells from bone marrow was associated with decreased patient survival (P=0.04), whereas the survival difference according to detection of CK-positive cells on primary screening was not statistically significant. In conclusion, viable tumour cells can be detected in the bone marrow of breast cancer patients. Their proliferative potential could be predictive of outcome and deserves further investigation.     

Effect of different cytokines on mammaglobin and maspin gene expression in normal leukocytes: possible relevance to the assays for the detection of micrometastatic breast cancer

In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of metastatic disease and monitoring of the efficacy of systemic therapy. These objectives remain elusive mainly due to the lack of specific genetic markers for solid tumours. The use of surrogate tissue-specific markers can reduce the specificity of the assays and give rise to a clinically unacceptable false-positive rate. Mammaglobin (MAM) and maspin are two putative breast tissue-specific markers frequently used for detection of occult tumour cells in the peripheral blood, bone marrow and lymph nodes of breast cancer patients. In this study, it was evaluated whether MAM and maspin gene expression may be induced in the normal blood and bone marrow cells exposed to a panel of cytokines, including chemotactic factors (C5a, interleukin (IL)-8), LPS, proinflammatory cytokines (TNF-alpha, IL-1beta) and growth factors (IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor). The experimental data show that all cytokines included in the panel, except for IL-8, were able to induce maspin expression; on the contrary, MAM gene was never induced. These results suggest that MAM is more specific than maspin and that the possible interference of cytokines should be taken into account in interpreting molecular assays for detection of isolated tumour cells.     

Detection and clinical significance of disseminated tumour cells at diagnosis in bone marrow of children with localised rhabdomyosarcoma

Identification of patients with a poor prognosis for non-metastatic rhabdomyosarcoma (RMS) remains a clinical challenge. Prospective analysis for the presence of disseminated RMS cells in bone marrow at diagnosis, using immunocytochemistry, with MyoD1 and myogenin as markers, was carried out. Thirty-seven patients treated on RMS88 and RMS96 Italian protocols underwent staging investigations, and in addition marrow examination for occult tumour cells. All patients had negative marrow involvement using cytomorphology, but 10/37 were positive with immunostaining. With a median follow-up of 46 months (range, 12-115), 7 patients had died and 30 were disease-free. Overall survival probability was 92% in patients with no occult marrow infiltration, 47% with occult marrow infiltration (P=0.001); event-free survival probability was 89% in the former and 50% in the latter (P=0.01). Disseminated tumour cells are indicative of disease spread and are significantly linked to recurrence at distant sites and poorer outcome. Marrow examination at diagnosis using immunocytochemistry may be an additional tool to modulate treatment.     

Clinical relevance and biology of circulating tumor cells

Most breast cancer patients die due to metastases, and the early onset of this multistep process is usually missed by current tumor staging modalities. Therefore, ultrasensitive techniques have been developed to enable the enrichment, detection, isolation and characterization of disseminated tumor cells in bone marrow and circulating tumor cells in the peripheral blood of cancer patients. There is increasing evidence that the presence of these cells is associated with an unfavorable prognosis related to metastatic progression in the bone and other organs. This review focuses on investigations regarding the biology and clinical relevance of circulating tumor cells in breast cancer.     

Detection of occult metastases in lung carcinomas: Progress and implications for lung cancer staging

The ability to detect occult regional and systemic metastases in patients with operable lung carcinoma could have a significant impact on the management of the disease. Here, we review the literature, including studies from our own laboratory, regarding the clinical significance of the presence of occult metastases in patients with lung cancer. The accumulated evidence strongly suggests that the detection of occult regional and systemic metastases is an important predictor of disease progression. The use of this method should be considered in the future design of lung cancer clinical trials, at the very least. The detection of occult metastases should have an impact on lung cancer mangement; to reflect this, we propose a change in the TNM staging system to indicate the presence or absence of occult regional (lymph node) and systemic (bone marrow) metastases. The proposed change is TNnMm, where n and m are occult nodal and bone marrow metastases status. J. Surg. Oncol. 1998;69:265–274. © 1998 Wiley-Liss, Inc.     

乳腺癌前哨淋巴结及骨髓中微转移的联合检测

目的：探讨乳腺癌患者联合进行前哨淋巴结（SLN）及骨髓微转移检测的可行性及临床价值。方法：^99mTc-SC作为示踪剂，进行前哨淋巴结活检，使用流式细胞仪检测前哨淋巴结中的微转移，并使用EMA和CK19的免疫组化染色，对腋窝淋巴结常规检查阴性的乳腺癌患者进行骨髓微转移的检测。结果：13例腋窝淋巴结常规HE检查阴性的患者中，流式细胞仪发现6例患者前哨林巴结中存在微转移（46．2％），SLN微转移的发生率与肿块大小相关（P＝0．027）。在腋窝淋巴结常规HE检查阴性的20例乳腺癌患者中，9例患者的骨髓中存在EMA或CK阳性的细胞（45％），骨髓微小转移与肿瘤ER（P＝0．045），OR（0＝0．010）的状态相关。10例同时进行骨髓及SLN微转移检测的患者，4例pN0(i )，4例pM0(i )，2例pN0(i )Mo(i )。结论：骨髓及淋巴结中的微转移可能与患者预后差相关。     

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.     

Characterization of disseminated tumor cells.

The most prominent secondary organs screened for the presence of occult disseminated tumor cells are regional lymph nodes and bone marrow. The current data suggest that micrometastatic cells represent a selected population of dormant cancer cells, which still express a considerable degree of heterogeneity. The analysis of micrometastatic cells will open a new avenue to assess the molecular determinants of both early tumor cell dissemination and subsequent outgrowth into overt metastases. Moreover, identifying therapeutic target structures (e.g., HER2), monitoring the elimination of bone marrow micrometastases, and assessing treatment-resistant tumor cell clones may help in understanding the current limitations of adjuvant systemic therapy. This review summarizes the current knowledge on the biological characteristics of micrometastatic cancer cells in bone marrow and lymph nodes of cancer patients.     

Prognostic significance of micrometastasis in non-small-cell lung cancer

Accurate staging of non-small-cell lung cancer (NSCLC) determines prognosis and facilitates decisions regarding treatment options. Unfortunately, even after an apparently complete resection in patients with stage I disease, the recurrence rates range from 25% to 50%, and overall survival is not encouraging. One possible reason for this may be that those patients with a poor outcome actually have more extensive disease, with occult locoregional and/or distant metastasis than originally identified by routine pathologic staging techniques. There is now a sizable body of literature on the detection and possible prognostic role of occult disease in lung cancer. The majority of these studies are based on immunohistochemical analysis of lymph nodes and/or bone marrow, but a handful of studies use molecular approaches. The purpose of this review is to summarize and critique the current literature on occult tumor cell spread to lymph nodes and bone marrow in patients with NSCLC. Based on this literature, we believe that the prognostic significance of bone marrow micrometastasis remains unclear. However, the majority of studies indicate that occult lymph node disease is associated with a poor outcome. Thus, our ability to detect individual tumor cells could result in more accurate staging of NSCLC in patients and would potentially lead to the development of novel therapies, as well as influence decisions regarding the use of appropriate multimodality treatment strategies, the choice of surgical technique, and extent of dissection. As data accumulate, the presence or absence of occult nodal involvement should probably be considered at the next revision of the staging system for NSCLC.     

Review: Biological relevance of disseminated tumor cells in cancer patients

The prognosis of cancer patients is largely determined by the occurrence of distant metastases. In patients with primary tumors, this relapse is mainly due to clinically occult micrometastasis present in secondary organs at primary diagnosis but not detectable even with high resolution imaging procedures. Sensitive and specific immunocytochemical and molecular assays enable the detection and characterization of disseminated tumor cells (DTC) at the single cell level in bone marrow (BM) as the common homing site of DTC and circulating tumor cells (CTC) in peripheral blood. Because of the high variability of results in DTC and CTC detection, there is an urgent need for standardized methods. In this review, we will focus on BM and present currently available methods for the detection and characterization of DTC. Furthermore, we will discuss data on the biology of DTC and the clinical relevance of DTC detection. While the prognostic impact of DTC in BM has clearly been shown for primary breast cancer patients, less is known about the clinical relevance of DTC in patients with other carcinomas. Current findings suggest that DTC are capable to survive chemotherapy and persist in a dormant nonproliferating state over years. To what extent these DTC have stem cell properties is subject of ongoing investigations. Further characterization is required to understand the biology of DTC and to identify new targets for improved risk prevention and tailoring of therapy. Our review will focus on breast, colon, lung, and prostate cancer as the main tumor entities in Europe and the United States.     

Oestrogen receptor status of primary breast carcinomas and their metastases. Relation to pattern of spread and survival after recurrence

Immunohistochemical antibody techniques for detection of oestrogen receptors (ER) were applied to formalin fixed, paraffin embedded sections from 62 primary breast cancers, the metastases of their original regional lymph nodes (29 cases), bone marrow carcinosis (43 cases) and liver metastases (20 cases). Forty per cent of the primary tumours and 31% of the regional lymph node metastases were ER positive; in contrast, less than 20% of liver and bone marrow metastases were ER positive. The ER status of regional lymph node metastases was concordant with that of the primary tumour in 90% of the cases. The concordance rate was 75% for liver metastases and 58% for bone metastases. Patients with ER positive primary tumours had recurrence significantly more often in bone; ER negative tumours recurred more often in the liver. The survival after recurrence (SAR) was significantly related to the ER status of the primary tumour and to that of the regional lymph node metastases. In contrast, the SAR was not associated with the ER status of bone marrow carcinosis or liver metastases. Cox analyses showed that age and ER status of the primary tumour were the most important independent prognostic factors compared to other clinical, therapeutic, pathoanatomical and biochemical features. The study supports the hypothesis that tumour cell clones with different ER content are selected and adapted to grow in various anatomical sites. Moreover, the ER status of the primary tumour seems to be more important for the prognosis than the ER status of bone and liver metastases.     

The presence of bone marrow cytokeratin-immunoreactive cells does not predict outcome in gastric cancer patients

The independent prognostic significance of isolated tumour cells in bone marrow is still a matter of debate. This study evaluated the possible association of bone marrow micrometastases with tumour progression and prognosis in patients affected by gastric cancer. Bone marrow aspirates from both iliac crests were obtained from 114 consecutive patients operated on for gastric cancer. The specimens were stained with monoclonal antibody CAM 5.2 which reacts predominantly with cytokeratin filaments 8 and 19. Among 114 cases analysed, 33 cases (29%) had cytokeratine-positive cells in the bone marrow. There was no significant relationship between the presence of bone marrow micrometastases and site, depth of tumour invasion, lymph node metastases, presence of metastases. Patients with cytokeratine-positive cells had a trend towards a diffuse type histology (P=0.06). Among the 88 curatively resected patients, median survivals were 40 months and 36 months for cytokeratine-negative and cytokeratine-positive subsets respectively (P=0.9). Recurrence of the disease was observed in 39 cases (44.3%); 11 of 24 (45.8%) in the cytokeratine-positive subset and 28 of 64 (43.7%) in the cytokeratine-negative subset. In conclusion in our experience the presence of cytokeratine-positive cells in the bone marrow of curatively resected gastric cancer patients did not affect outcome and its independent prognostic significance remains to be proven before its official acceptance in the TNM classification.     

Implications of occult metastatic cells for systemic cancer treatment in patients with breast or gastrointestinal cancer.

The early and clinically occult spread of viable tumour cells to the organism is becoming acknowledged as a hallmark in cancer progression, since abundant clinical and experimental data suggest that these cells are precursors of subsequent distant relapse. Using monoclonal antibodies against epithelial cytokeratins or tumour-associated cell membrane glycoproteins, individual carcinoma cells can be detected in cytological bone marrow preparations at frequencies of 10(-5) to 10(-6). Prospective clinical studies have shown that the presence of such immunostained cells in bone marrow is prognostically relevant with regard to relapse-free and overall survival, even in malignancies that do not preferentially metastasise to bone. As current treatment strategies have resulted in a substantial improvement of cancer mortality rates, it is noteworthy to consider the intriguing options of immunocytochemical screening of bone marrow aspirates for occult metastatic cells. Besides improved tumour staging, such screening offers opportunities for guiding patient stratification for adjuvant therapy trials, monitoring response to adjuvant therapies (which, at present, can only be assessed retrospectively after an extended period of clinical follow-up), and specifically targeting tumour-biological therapies against disseminated tumour cells. The present review summarises the current data on the clinical significance of occult metastatic cancer cells in bone marrow.     

Detection of hematogenic and lymphogenic tumor cell dissemination in patients with medullary thyroid carcinoma by cytokeratin 20 and preprogastrin-releasing peptide RT-PCR

Despite an extensive surgical approach only 50% of the patients with medullary thyroid carcinoma (MTC) are biochemically cured. The failure to cure a larger number of patients is a result of the early dissemination of MTC. The present study evaluates two RT-PCR based assays for the detection of disseminated tumor cells in blood, bone marrow and lymph node samples of patients with MTC. Frozen tissue and blood samples of 19 patients with MTC and 61 cervical lymph nodes of these patients were obtained intraoperatively during thyroidectomy and lymphadenectomy. Preoperative bone marrow samples were obtained from 8 patients with MTC. An expression of CK20 and preproGRP was found in all MTC tissue samples. Using CK20-PCR, disseminated MTC cells were detected in 67% of the cervical lymph nodes of patients with MTC, compared to 72% involved lymph nodes, detected by preproGRP-PCR. In 16 of 61 nodes (26%) each PCR-system detected disseminated tumor cells in histologically tumor-free lymph nodes. Disseminated tumor cells were detected with CK20-PCR and preproGRP in 5 of 18 (28%) preoperative blood samples, each. The detection of a hematogenic tumor cell dissemination by preproGRP correlated significantly with the tumor stages (p = 0.019). Circulating MTC cells were found in 3 of 8 bone marrow samples with CK20-PCR, compared to 1 of 8 samples with preproGRP-PCR. Both PCR assays are highly sensitive to detect disseminated MTC cells in blood, bone marrow and lymph node samples. Our results of disseminated MTC cells in 26% of histologically tumor-free cervical lymph nodes and in 28% of the blood samples of patients with MTC might therefore explain the low biochemical cure rates.