Isolation and characterization of circulating tumor cells (CTCs) in patients suffering from a variety of different cancers have become hot biomarker topics. In this study, we evaluated the prognostic value of CTCs in pancreatic cancer.

Initial literature was identified using Medline and EMBASE. The primary data were hazard ratios (HRs) with 95% confidence intervals (CIs) of survival outcomes, including overall survival (OS) and progression free survival/recurrence free survival (PFS/RFS).

A total of 9 eligible studies were included in this meta-analysis, published between 2002 and 2013. The estimated pooled HR and 95%CI for OS for all studies was 1.64 (95%CI 1.39-1.94, p<0.00001) and the pooled HR and 95%CI for RFS/DFS was 2.36 (95%CI 1.41-3.96, p<0.00001). The HRs and 95%CIs for OS and RFS/ DFS in patients before treatment were 1.93 (95%CI 1.26-2.96, p=0.003) and 1.82 (95%CI 1.22-2.72, p=0.003), respectively. In patients receiving treatment, the HRs and 95%CI for OS and RFS/DFS were 1.37 (95%CI 1.00- 1.86, p=0.05) and 1.89 (95%CI 1.01-3.51, p=0.05), respectively. Moreover, the pooled HR and 95%CI for OS in the post-treatment group was 2.20 (95%CI 0.80-6.02, p=0.13) and the pooled HR for RFS/DFS was 8.36 (95%CI 3.22-21.67, p<0.0001).

The meta-analysis provided strong evidence supporting the proposition that CTCs detected in peripheral blood have a fine predictive role in pancreatic patients especially on the time point of post-treatment.

Occult neoplastic cells (ONCs) are the tumor cells floating in the lymph node sinuses, distant from the primary tumor, and supposed to be one of most reliable marker of prognosis.

We report here the case of a 52-year-old woman with a gastric cancer associated by numerous ONCs.

Postoperative examination of the stomach disclosed an advanced, poorly differentiated adenocarcinoma with frequent lymph node metastases. In addition to ONCs and occasional micrometastases, focal aggregates of ONCs, one of the possible intermediate lesions between the ONCs and the usual metastases, are also observed.

In the present case, at least some of ONCs seem to form the microaggregates of tumor cells in lymph nodes, anchor in the sinuses, and grow up to the large tumorous lesion. Even if most of the ONCs were trapped and disappeared under the influence of tumor immunity, the detection of ONCs could be one of the reliable clues to estimate the prognosis.

Despite the modest improvements in patient survival from colorectal cancer in the last few decades, the overall five-year survival rate remains at 40 to 45 percent. Surgical resection is the mainstay of treatment for colorectal cancer; however, nearly one-half of all patients who undergo a potentially curative resection will relapse because of undetected micrometastasis. The fact that the overall survival rate remains poor strongly suggests that the dissemination of these cells occurs early in the disease process and emphasizes the need for finding feasible diagnostic methods with sufficient sensitivity and specificity. The most commonly used technique for the detection of nucleic acid material of disseminated tumor cells is the polymerase chain reaction. We critically review the literature on DNA and messenger ribonucleic acid molecular markers that have been used for the detection of circulating tumor cells in the peripheral blood of patients with colorectal cancer and other solid tumors as appropriate for comparison. The cytokeratins, particularly cytokeratin 19 and cytokeratin 20, are the most investigated prognostic markers, but even for these questions remain about their clinical value, and hence most recent studies are utilizing a combination of factors. There is an urgent need for standardized isolation and analysis techniques to be adopted thus allowing large-scale, appropriately controlled, multicenter trials to be undertaken on the most promising candidate markers.

This investigation intended to study the unspecific background to be expected in normal bone marrow (BM), comparing three well recognized protocols for immunocytochemical detection of disseminated carcinoma cells. The interlaboratory variation in screening and evaluation of stained cells was analyzed and different screening methods were compared.

BM mononuclear cells (BM MNC) from 48 normal BMs were immunostained in parallel by three participating laboratories. The protocols, based on three different anti-cytokeratin antibodies, have all been in common use for detection of disseminated carcinoma cells: the A45-B/B3 protocol (Hamburg), the CK2 protocol (Augsburg) and the AE1AE3 protocol (Oslo). For all protocols, the immunostained cells were visualized by the same alkaline-phosphatase (AP) detection system (APAAP) followed by detection of the cells by manual screening and by two different automated screening systems (ACIS from Chromavision and MDS1 from Applied Imaging). Detected AP-visualized cells were morphologically classified into unambiguous hematopoietic (Uhc) and questionable cells (Qc, potentially interpreted as tumor cells).

Seven of 48 BMs (15%) harbored > or = 1 AP-visualized cell(s) among 1 x 10(6) BM MNC, both for the A45-B/B3- and for the AE1AE3 protocol, while for CK2 a higher proportion of BMs (21 BMs; 44%) harbored AP-visualized cells (P < 0.01, McNemar's test). The number of Qc was, for all protocols, 1 log lower than the total number of AP-visualized cells. On average, the frequency of Qc was 0.04, 0.08, and 0.02 per 10(6) BM MNC with A45-B/B3, CK2 and AE1AE3, respectively, and the number of Qc-positive BMs 1, 4, and 1. The MDS1 screening sensitivity was similar to manual screening, while ACIS detected fewer cells (P < 0.001, McNemar's test).

All protocols resulted in AP-visualization of occasional hematopoietic cells. However, morphological classification brings the specificity to a satisfactory high level. Approximately 10% of AP-visualized cells were categorized "questionable". The CK2 protocol turned out less specific than the A45-B/B3 and AE1AE3 protocols.

Despite improvements in locoregional treatment of head and neck squamous cell carcinoma (HNSCC), local and distant failure rates remain high. The strongest prognostic indicator of HNSCC is the presence of lymph node metastases in the neck, but the value of this indicator has limitations when using for the individual patient. The presence of micrometastatic cells in bone marrow has been shown to be a putative prognostic indicator in HNSCC and other epithelial malignancies, which might allow more accurate staging and selection of patients for whom adjuvant or experimental therapy is recommended. The gene encoding the E48 antigen is selectively expressed by HNSCC, and the detection of E48 transcripts in bone marrow by reverse transcription-polymerase chain reaction (RT-PCR) presumably represents the presence of micrometastatic cells. The purpose of this study was to determine the association between the presence of micrometastatic cells in bone marrow of HNSCC patients and clinical outcome.

A total of 162 patients treated surgically for primary HNSCC underwent a single bone marrow aspiration from the upper iliac crest for detection of micrometastatic cells using E48 RT-PCR. In total, 139 patients were evaluable. The primary statistical endpoints were disease-free survival and distant metastasis-free survival. In addition, bone marrow samples of 30 noncancer controls were evaluated.

E48 RT-PCR indicated the presence of micrometastatic cells in the bone marrow in 56 of 139 (40%) of the HNSCC patients and 0 of 30 of the noncancer controls (P < 0.0001). The presence of micrometastatic cells had no significant influence on disease-free survival or distant metastasis-free survival for the whole group of HNSCC patients (P = 0.1460 and P = 0.2912, respectively). For patients with >or=2 lymph node metastases, however, the presence of micrometastatic cells was associated with a poor distant metastasis-free survival (P = 0.0210).

The presence of micrometastatic cells in bone marrow of HNSCC patients with >or=2 lymph node metastases is correlated with a poor distant metastasis-free survival. In this subgroup of HNSCC patients, E48 RT-PCR seems to be a valuable tool to identify patients who are at increased risk for development of distant metastases and therefore might benefit from experimental adjuvant systemic therapy.

Breast cancer is noted for long periods of tumor dormancy and metastases can occur many years after treatment. Adjuvant chemotherapy is used to prevent metastatic recurrence but is not always successful. As a model for studying mechanisms of dormancy, we have used two murine mammary carcinoma cell lines: D2.0R/R cells, which are poorly metastatic but form metastases in some mice after long latency times, and D2A1/R cells, which form more numerous metastases much earlier. Previously we identified a surprisingly large population of dormant but viable solitary cells, which persisted in an undivided state for up to 11 weeks after injection of D2.0R/R cells. Dormant cells were also detected for D2A1/R cells, in a background of growing metastases. Here we used this model to test the hypothesis that dormant tumor cells would not be killed by cytotoxic chemotherapy that targets actively dividing cells, and that the late development of metastases from D2.0R/R cells would not be inhibited by chemotherapy that effectively inhibited D2A1/R metastases. We injected mice with D2A1/R or D2.0R/R cells via a mesenteric vein to target liver. We developed a doxorubicin (DXR) treatment protocol that effectively reduced the metastatic tumor burden from D2A1/R cells at 3 weeks. However, this treatment did not reduce the numbers of solitary dormant cells in mice injected with either D2A1/R or D2.0R/R cells. Furthermore, DXR did not reduce the metastatic tumor burden after an 11-week latency period in mice injected with D2.0R/R cells. Thus, apparently effective chemotherapy may spare non-dividing cancer cells, and these cells may give rise to metastases at a later date. This study has important clinical implications for patients being treated with cytotoxic chemotherapy.

The aim of this study was to determine the influence and significance of different aspiration sites and the number of mononuclear cells (MNC) analyzed on the frequency of isolated tumor cell (ITC) detection by immunocytochemistry (ICC) in BM aspirates from breast-cancer patients.

BM aspirates were collected from the two anterior and two posterior crests just prior to primary surgery. The BM was processed separately from the anterior and the posterior crests, and cytospins (2 x 10(6) MNC) were prepared for ICC examination. The remaining cells were pooled, followed by cytospin preparation and ICC analysis (2 x 10(6) MNC/test). In addition, a fraction of the pooled cells were further processed by negative immunomagnetic selection, for enrichment of ITC. Out of 100 patients selected, 97 were further analyzed.

The ICC examination from the separate crests revealed 37 positive BMs from the anterior iliac crest and 30 positive from the posterior crest. Twenty-one of the samples were positive at both sides. Five patients had 10 or more ITCs detected. In these, an unequal distribution of ITCs between the sides was observed, but in favor of neither. ICC analysis of 2, 4 and 6 x 10(6) MNC revealed respectively 22, 46 and 52 positive BMs out of the 97 analyzed. These results were correlated to the clinical outcome after a median 43 months follow-up. Thirteen of the patients underwent systemic relapse. Analyzing 2 x 10(6) MNC by ICC, 27.3% of the BM+ patients developed systemic disease, compared with 9.3% of the BM+ patients (P = 0.0056, log rank test). Analyzing 6 x 10(6) MNC reduced the correlation between ITC in BM and clinical outcome.

No significant difference in the detection rate of ITCs from the anterior and the posterior iliac crests was found, although the distribution of ITCs did show a great variability. Analyzing a higher number of BM cells increased the number of positive BM specimens detected. However, this increased detection rate reduces the prediction by ICC of early systemic relapse.

In curatively resected gastric cancer, the incidence of distant relapse is as high as 30%. Although the most important factor contributing to the local control of the tumor is the microscopic tumor-free margin of the surgical resection, the occurrence of distant metastases is in many cases due to preoperative or perioperative tumor cell dissemination. In addition to the established TNM staging system, disseminated tumor cells may serve as independent prognostic factors influencing patient outcome after curative surgery. Basically, in gastric cancer three compartments have been identified in which single tumor cells may be shed: lymph nodes, peritoneal cavity, and bone marrow. Assessment of resected regional lymph nodes with monoclonal antibodies directed against cytokeratin antigens leads to an upstaging in comparison with conventional histology. Nodal micrometastases detected by immunohistochemistry result in an upstaging of up to 36% of patients. However, their prognostic significance remains controversial. Local dissemination of tumor cells in the peritoneal cavity determines the outcome in advanced gastric cancer and diffuse-type carcinoma. Patients with negative peritoneal washings seem to have a more favorable prognosis. Moreover, with the use of these diagnostic tools, patient subpopulations may be identified which profit from intraperitoneal therapy regimens. Diffuse hematogenous tumor cell dissemination into the bone marrow has been shown to be a prognostic factor in several studies. In our own population of 180 gastric cancer patients, bone marrow cells were screened immunohistochemically with a monoclonal antibody directed against cytokeratin 18 (CK18). In 95 patients (53%), CK2-posititve cells were detected. In a multivariate analysis, the independence of the presence of three or more disseminated tumor cells per 10(6) mononuclear cells was proven to be a prognostic factor in patients with intestinal-type tumors, pT1/2 status, and pN0 status. In conclusion, the TNM status only partially reflects the actual extent of systemic disease in patients with resected gastric cancer. The assessment of minimal residual disease is valuable in estimating the prognosis in many patients. In the future, staging systems will have to not only include TNM data but also provide specific information on biological properties of residual cancer cells in order to establish more exact prognostic estimates and provide patients with an individually tailored multimodal treatment.

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.

The use of automated microscopy has reached the maturity necessary for its routine use in the clinical pathology laboratory. In the following study we compared the performance of an automated microscope system (MDS) with manual method for the detection and analysis of disseminated tumor cells present in bone marrow preparations from breast carcinoma patients. The MDS System detected rare disseminated tumor cells among bone marrow mononuclear cells with higher sensitivity than standard manual microscopy. Automated microscopy also proved to be a method of high reproducibility and precision, the advantage of which was clearly illustrated by problems of variability in manual screening. Accumulated results from two pathologists who had screened 120 clinical slides from breast cancer patients both by manual microscopy and by use of the MDS System revealed only two (3.8%) missed by the automatic procedure, whereas as many as 20 out of 52 positive samples (38%) were missed by manual screening.

To study bone marrow micrometastases from colorectal cancer patients for the presence of K-ras mutations and to compare their genotype with that of the corresponding primary tumor.

Bilateral iliac crest aspiration was performed in 51 patients undergoing surgery for colorectal cancer, and bone marrow micrometastases were detected by immunohistochemistry. The presence of K-ras mutations was determined by single-strand conformation polymorphism analysis on both primary tumors and paired bone marrow samples and was confirmed by sequencing.

In six patients with primary tumor mutations, it was possible to amplify a mutated K-ras gene also from the bone marrow sample. In three of those patients the pattern of K-ras mutations differed between both samples, in two patients the mutation was identical between the bone marrow and its primary tumor, and in one patient the same mutation plus a different one were found. Fifteen of 17 K-ras mutations found in primary tumors were located in codon 12, whereas in bone marrow, five of seven mutations were found in codon 13 (P =.003).

Our results demonstrate that, at least for K-ras mutations, disseminated epithelial cells are not always clonal with the primary tumor and they question the malignant genotype of bone marrow micrometastases. They also indicate that different tumoral clones may be circulating simultaneously or sequentially in the same patient. Analysis of the type of mutations suggests that cell dissemination might be an early event in colorectal carcinogenesis.

Tumor progression after curative resection of gastrointestinal carcinomas is probably caused by pre- or intraoperative tumor cell dissemination. Disseminated tumor cells are generally detected by immunohistochemistry- or PCR-based molecular-biology methods. A consensus on which is the most adequate detection method has not yet been found, which makes the comparison of data difficult. The prognostic relevance of disseminated cells has been shown, at least in part, for esophageal, gastric, pancreatic, and colonic cancer. The data regarding hepatocellular cancer is conflicting. This article gives a critical review of tumor cell detection in gastrointestinal cancer.