bromochloroacetic-acid and Bone-Marrow-Neoplasms

The presence of tumor cells in the bone marrow of primary breast cancer patients at surgery has been shown to be an independent prognostic indicator of relapse. Tumor cells have been detected either directly, using immunocytochemical staining, or indirectly, using reverse transcription-polymerase chain reaction (RT-PCR). Studies have been initiated to determine whether the presence of disseminated cells can be monitored during the therapy of patients with primary breast cancer, and thus potentially be used to predict relapse before overt metastases are detectable. Studies are also ongoing to improve methods of detection, such as immunobead enrichment followed by staining and real-time RT-PCR, and to find alternative markers for the disseminated cells. Studies of patients with overt metastases have shown that there is a large tumor load in the peripheral blood and that this predicts overall survival. This article reviews the published literature on studies carried out in both primary and metastatic breast cancer patients, the methodologies and markers used, and improvements in detection methodologies that are being investigated including real-time RT-PCR, novel markers, enrichment and automated image analysis.

Topics: Biomarkers, Tumor; Bone Marrow; Bone Marrow Neoplasms; Breast Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Reverse Transcriptase Polymerase Chain Reaction

Topics: Biomarkers, Tumor; Bone Marrow Cells; Bone Marrow Neoplasms; Breast Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm

Despite an increasing molecular-genetic understanding of the development of malignant epithelial neoplasias, the frontline therapy for patients with carcinomas is still surgery. Systemic adjuvant treatments such as chemotherapy or immunotherapy have had limited success perhaps because they are based on analysis of the primary tumour or on cell lines derived from metastasis. However, the characteristics of systemically disseminated tumour cells can be very different from that of the primary tumour or end-stage metastasis. Consequently, there is a need to study the evolution and nature of systemic cancer directly in order to identify new target structures for therapy present on the potential precursors of metastasis--the disseminated tumour cells.

Topics: Animals; Bone Marrow; Bone Marrow Neoplasms; Disease Progression; DNA Mutational Analysis; Epithelial Cells; Humans; Keratins; Neoplasm Metastasis; Neoplasm, Residual; Neoplasms; Tumor Escape

Topics: Bone Marrow; Bone Marrow Examination; Bone Marrow Neoplasms; Breast Neoplasms; Colorectal Neoplasms; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Lymphatic Metastasis; Neoplastic Cells, Circulating; Ovarian Neoplasms; Prognosis; Prospective Studies; Randomized Controlled Trials as Topic; Risk Factors; Stomach Neoplasms; Uterine Cervical Neoplasms

Data are emerging about the prognostic relevance of occult metastatic cells in the bone marrow of patients with various solid tumors. Discrepancies among different studies on the prognostic relevance of isolated tumor cells may be caused by tumor cell heterogeneity and the use of different immunoassays. There is increasing evidence that validated anticytokeratin antibodies (e.g., A45-B/B3) represent the present standard for the detection of isolated tumor cells. This immunocytochemical assay allows the identification of patients with occult tumor cell dissemination that cannot be identified by conventional screening methods in tumor staging. According to recent studies, these patients are at higher risk for subsequent development of distant metastases and might therefore benefit from early systemic therapy. At advanced stages of the disease, the micrometastatic tumor load after adjuvant therapy, or at the time of emerging recurrences, appears to reflect the tumor's ability to progress. Therapeutic monitoring and cell-cycle independent antibody-based therapy are among possible implications of this new, promising diagnostic tool. The present review also focuses on state of the art, reliable detection methods of occult metastatic cells in the bone marrow of breast cancer patients and on the prognostic relevance of these cells at different stages of the disease.

Topics: Antigens, Neoplasm; Biopsy, Needle; Bone Marrow Neoplasms; Breast Neoplasms; Clinical Trials as Topic; Female; Humans; Keratins; Neoplasm Staging; Neoplastic Cells, Circulating

In this study we have validated the feasibility of detecting disseminated tumor cells (DTC) by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Bone marrow samples from a large cohort of patients with breast cancer were analyzed for the presence of DTC by immunocytochemistry (ICC) or a molecular-based method.. Bone marrow samples were collected from 170 patients with breast cancer with stage I-IV disease before the initiation of any local or systemic treatment. Staining for cytokeratin (CK)-positive cells was performed with the Epimet kit. Disseminated tumor cells were also quantified by measuring relative gene expression for CK19 and mammaglobin (MAM) using a quantitative RT-PCR detection method. The mean follow-up time was 30 months. Kaplan-Meier analysis was used for predicting overall survival.. Despite an excellent quantitative correlation and qualitative concordance between ICC and RT-PCR, survival analysis suggested an improved prognostic significance of DTC as detected by quantitative RT-PCR. Univariate survival analysis computed a relative risk of death of 2.87 for women with ICC-positive cells in the bone marrow, as compared with those without positive cells. The relative risk for women with RT-PCR-positive bone marrow was even higher: 3.5 (CK19) and 3.39 (MAM). In multivariate analysis, bone marrow CK19 was a stronger prognostic factor than bone marrow ICC.. Reverse-transcriptase polymerase chain reaction-detected DTC is shown to be prognostically significant in untreated patients with breast cancer. Furthermore, it seems to be a more sensitive method for detecting DTC in bone marrow samples when compared with ICC.

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow Neoplasms; Breast Neoplasms; Feasibility Studies; Female; Humans; Immunohistochemistry; Keratins; Mammaglobin A; Middle Aged; Neoplasm Proteins; Neoplastic Cells, Circulating; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Uteroglobin

Micrometastatic disease in bone marrow is of prognostic significance in colorectal cancer patients. However, detection rates of standard immunocytology are relatively low. We used magnetic activated cell sorting (MACS), a highly sensitive method, to increase detection rates and correlated the presence of cytokeratin (CK)-expressing cells with clinical parameters.. Bone marrow was obtained from 51 consecutive patients with newly diagnosed colorectal adenocarcinoma who underwent primary surgery and 18 control subjects. International Union Against Cancer (UICC) stage I disease was diagnosed in 11 patients, stage II disease was diagnosed in 14 patients, stage III disease was diagnosed in 12 patients, and stage IV disease was diagnosed in 14 patients. CK-positive cells were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to CK 7 and 8.. CK-positive cells were found in 33 (65%) patients and were absent in 18 (35%). Four of 11 (36%) patients with UICC stage I disease, nine of 14 (64%) with stage II diease, eight of 12 (67%) with stage III disease, and 12 of 14 (86%) with stage IV disease were CK-positive. Epithelial cells were more frequently found in pT3/4 (72%) than in pT1/2 (36%) tumors (P =.026), but there was no difference for lymph node status. CK-positive patients had a higher chance for elevated carcinoembryonic antigen (85% v 15%, P = NS) and CA 19-9 levels (92% v 8%, P =.019). There were no significant differences in CA 72-4, sex, age, tumor grading, or tumor localization regarding the presence of CK-positive cells. All control subjects were CK-negative.. In searching for micrometastases in colorectal cancer patients, we have achieved high detection rates by using MACS. The presence of these cells correlated significantly with tumor stage, tumor extension, and the tumor marker CA 19-9.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Bone Marrow Neoplasms; Colorectal Neoplasms; Female; Humans; Immunomagnetic Separation; Keratins; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Sensitivity and Specificity

An ultrasensitive tumor enriched flow-cytometric assay was used to determine its feasibility in detection of isolated tumor cells (ITC) in bone marrow (BM) of patients with breast cancer.. Epithelial cells were removed by magnetic microbeads conjugated with an anti-cytokeratin 7/8 monoclonal antibody to enrich tumor cells in BM samples. A specific gate for MCF-7 breast cancer cells (gate(MCF-7 cells)) was also taken into consideration in addition to a gate including all enriched BM cells (gate(enriched BM cells)) in flow-cytometric analysis to enhance the specificity of the method.. Nineteen patients with stage I/II were evaluated. Ten patients (53%) were found to have cytokeratin positive (CK(+)) cells according to the gate(enriched BM cells) whereas 6 patients (32%) had CK(+) cells when the gate(MCF-7 cells) was taken into account.. New strategies in nonmorphological ultrasensitive techniques might be useful to categorize patients with ITCs having different tumor morphology and characteristics.

Topics: Adult; Aged; Antibodies, Monoclonal; Bone Marrow Neoplasms; Breast Neoplasms; Feasibility Studies; Female; Flow Cytometry; Humans; Immunomagnetic Separation; Keratins; Middle Aged; Neoplastic Cells, Circulating; Sensitivity and Specificity; Tumor Cells, Cultured

The detection of isolated tumor cells (ITC) in the bone marrow of patients with epithelial malignancies is an independant prognostic factor for several entities as breast cancer, colorectal cancer or non-small lung cancer. However, with conventional immunocytology using Ficoll density gradient and APAAP staining, only a small proportion of the bone marrow samples can be scanned for cytokeratin-positive (CK+) cells. To improve detection rates, we evaluated the enrichment of ITC by magnetic activated cell sorting (MACS) compared to regularly stained cytospins. Recovery experiments with a CK+ breast cancer cell line (SKBR3) were performed to calculate the MACS enrichment rate. Bone marrow was obtained by aspiration from 20 patients with carcinomas of epithelial origin and from 17 controls. ITC were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to cytokeratin 7 and 8. MACS of SKBR3 seeded in peripheral blood revealed average recovery rates of 62% and 48% and average enrichment factors of 104-fold and 8139-fold of the CK+ cells after one and after two separations, respectively. After immunomagnetic enrichment, CK+ cells were detected in 16 of 20 (80%) cancer patients, whereas only 7 (35%) patients showed CK+ cells without magnetic enrichment (P = 0.002). Ten of twelve (83%) patients with metastatic disease (stage M1) and six of eight (75%) patients without any overt metastases (M0) had CK+ cells in their bone marrow. None of the negative controls showed any CK+ cells. Enrichment with magnetically labeled anti cytokeratin antibodies increases the detection rate of epithelial cells in bone marrow of cancer patients compared to immunocytology.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Biomarkers, Tumor; Bone Marrow Neoplasms; Breast Neoplasms; Carcinoma; Carcinoma, Non-Small-Cell Lung; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Magnetics; Male; Middle Aged; Prospective Studies; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The presence of cytokeratin 18-positive cells in bone marrow correlates with conventional risk factors in many tumors. We examined whether this was also valid for localized or lymphatically spread prostate cancer.. Immediately before radical prostatectomy, bone marrow aspirates from both sides of the iliac crest were taken from 287 patients. The presence of cells containing cytokeratin 18 was interpreted as micrometastasis.. In patients with negative lymph nodes (n = 219), conventional risk factors (Gleason score, pathologic stage, ploidy, and preoperative prostate-specific antigen) did not correlate with the preoperative detection of cells containing cytokeratin 18. There was also no correlation with lymph node metastases. Furthermore, there was no interdependency between the preoperatively detected number of cells and the established risk factors.. We assume the presence of epithelial cells in bone marrow to be an independent parameter, the clinical importance of which must be substantiated by further studies.

Topics: Adenocarcinoma; Aged; Bone Marrow Neoplasms; Flow Cytometry; Humans; Keratins; Male; Middle Aged; Ploidies; Prognosis; Prostatectomy; Prostatic Neoplasms; Risk Factors

Despite current advances in antibody-based immunotherapy of breast and colorectal cancer, we have recently shown that the actual target cells (e.g., tumor cells disseminated to bone marrow) may express a heterogeneous pattern of the potential target antigens. Tumor antigen heterogeneity may therefore represent an important limitation of the efficacy of monospecific antibody therapy. To measure the efficacy of such a monospecific approach, we analyzed the elimination of tumor cells coexpressing the epithelial cell adhesion molecule (EpCAM) under therapy with murine monoclonal antibody 17-1A (Edrecolomab) directed against EpCAM. In bone marrow aspirates from 10 breast cancer patients with metastatic (n = 8) and locoregional recurrence (n = 2), tumor cells were identified with the antibody A45-B/B3 directed against the epithelial differentiation marker cytokeratin (CK) and simultaneously typed for EpCAM expression using the antibody 17-1A. Patients were treated with a single dose of 500 mg of Edrecolomab and monitored by bone marrow analyses before and at days 5-7 after antibody treatment. In all 10 patients, we assessed a marked reduction in the mean numbers of both CK+ cells (73 versus 15; P = 0.003, t test) and EpCAM+/CK+ cells (17 versus 1; P = 0.003, t test) per 10(6) bone marrow cells. Complete elimination of EpCAM+ cells was possible in four patients. We conclude that Edrecolomab can be used in breast cancer patients to target isolated EpCAM+/CK+ cancer cells. Using CK-based immunoassays, we reliably detected residual tumor cells in bone marrow and typed EpCAM expression. This allowed us to monitor the cytotoxic elimination of such cells after Edrecolomab application. Selection of EpCAM-/ CK+ tumor clones showed that further antibodies directed against tumor-associated antigens are warranted to improve the efficacy of monospecific approaches.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antibody Specificity; Antigens, Neoplasm; Biomarkers, Tumor; Bone Marrow Cells; Bone Marrow Neoplasms; Breast Neoplasms; Cell Adhesion Molecules; Cytotoxicity, Immunologic; Epithelial Cell Adhesion Molecule; Female; Growth Inhibitors; Humans; Immunohistochemistry; Keratins; Pilot Projects; Prospective Studies

The micrometastatic spread of tumour cells is usually missed by conventional diagnostic techniques, although this spread largely determines the prognosis of patients with primary epithelial cancers. By use of the monoclonal antibody, CK2, to epithelial cytokeratin component number 18 (CK18), individual disseminated carcinoma cells present in bone marrow of cancer patients can now be identified. In the present study, this approach has been applied to patients with virginal stage C adenocarcinoma of the prostate. Double-sided aspirates of iliac bone marrow from 24 of 44 evaluable patients (54.4%) exhibited between one and 38 CK18-positive cells per sample of 2 x 10(6) mononuclear cells. In 13 of these 24 positive patients, CK-positive cells were only detected in one of the two aspirates analysed. There was no statistically significant correlation between this finding and established risk factors, such as the volume and histological grade of the primary tumour or the concentration of prostate specific antigen and prostatic acid phosphatase in serum. The follow-up time is too short to provide meaningful data on the prognostic significance of isolated CK18-positive cells in bone marrow, which, however, has been recently demonstrated in other types of primary epithelial cancers. In conclusion, the presence of prostatic tumour cells in bone marrow might be interpreted as an indicator of the metastatic capacity of an individual primary tumour. The immunocytochemical detection of these cells may, therefore, be useful for increasing the precision of current tumour staging, and to monitor minimal residual cancer in an individual patient.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Biomarkers, Tumor; Bone Marrow Neoplasms; Humans; Immunoenzyme Techniques; Keratins; Male; Prostatic Neoplasms; Risk Factors

Behaviors of esophageal cancer are different according to the geographic distribution. The prevalence of bone marrow involvement in patients with esophageal cancer has been shown to be between 40% and 90%, but clinical correlation is unknown. The aim of this study is to determine the rate of bone marrow involvement in patients with esophageal cancer in the northeast of Iran and its relationship with clinicopathologic findings of the tumors.. A total of 43 patients with esophageal cancer, who were candidates for esophagectomy (without neo-adjuvant chemotherapy), were enrolled in this study from 2007 to 2009. Bone marrow samples derived from rib bone were stained with hematoxylin and eosin (H&E) to distinguish tumoral cells, and cytokeratin immunohistochemistry (CKIHC) was used to determine micrometastasis. The correlation of the results was studied with the histopathologic indices of primary tumor (T (tumor), N (node) and length of tumoral involvement and grading) as well as characteristics of the patients (sex and age).. The mean age was 64 (57-70) years and the M/F ratio was 2.9. As many as 38 patients (88.4%) had squamous cell carcinoma and five patients (11.6%) had adenocarcinoma. In nine cases (20.9%), the H&E test, and, in 13 cases (30.2%), the CKIHC evaluation was positive. Statistically, there was no relationship between the pathologic type and the stage of T with the H&E study and CKIHC test, respectively. On the other hand, a significant meaningful correlation was found between microscopic bone marrow involvement as well as mediastinal lymph node involvement and grade of the tumor.. Bone marrow involvement incidence was low in our geographic area. According to our study, bone marrow involvement in esophageal cancer is related to differentiation grade and mediastinal lymph node involvement.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Bone Marrow Neoplasms; Carcinoma, Squamous Cell; Cross-Sectional Studies; Esophageal Neoplasms; Esophagectomy; Female; Humans; Keratins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Grading; Neoplasm Micrometastasis; Neoplasm Proteins; Neoplasm Staging

Numerous studies have shown that the presence of clinically occult disseminated tumor cells (DTC's) in the bone marrow (BM) of breast cancer patients is associated with an unfavourable clinical outcome. Immunocytochemistry (ICC) remains the gold standard for their detection. While assays based on RT-PCR are available, they have not been used for routine detection of DTC's.. To assess the quality of the assay, we performed a direct comparison of DTC detection rates in a large cohort of 385 patients using both standardized ICC and real-time RT-PCR protocols. Correlation rates were assessed, and results were compared with clinical data.. A significant correlation between ICC and RT-PCR was observed (P < 0.01). Positivity rates were similar (both 35%) and the results of both methods agreed in 73% of cases (280/385).. We describe a real-time RT-PCR based protocol for DTC-detection that has been specifically designed for routine clinical laboratory use. As such, RT-PCR has the potential to become an alternative testing method for BM evaluation in breast cancer patients.

Topics: Bone Marrow Neoplasms; Breast Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Mass Screening; Neoplasm Staging; Sensitivity and Specificity

Bone marrow micrometastases (BMM) and sentinel lymph node (SLN) status are both prognostic factors in breast cancer (BRCa) patients (pts). A definitive relationship between the two has not yet been proven and the data available is controversial. Thus, a retrospective study was conducted to determine the relationship of BM status and SLN status in pts with early BRCa (T1/T2). All female pts with early BRCa (T1/T2) operated upon by a single surgeon were included in the study. Prior to surgery, all pts underwent bone marrow aspiration from the posterior superior iliac spine bilaterally. Subsequently, pts underwent SLN biopsy and definitive primary breast surgery. BM samples were examined by using a Cytokeratin Detection Kit using CAM 5.2 monoclonal antibody. All pts with BMM underwent repeat BM analysis 6 months after completing all treatments. Data was collected for SLN, BM, estrogen receptor/progesterone receptor (ER/PR), and human epidermal growth factor receptor 2 (Her-2/neu) status and analyzed using chi-square (chi (2)) analysis or Fischer's exact test. A total of 270 consecutive pts with early BRCa were studied. SLN mapping was successful in all pts. SLN metastases (mets) were detected in 28.9% (78/270) pts. Of the 270 pts, 77.0% (208/270) had T1 disease. BMM were detected in 9.6% (26/270) pts, of whom 69.2% (18/26) were found to have BMM unilaterally. BMM were detected in 11.5% (9/78) pts with SLN mets versus 8.9% (17/192) in pts with node-negative disease (p = 0.65). Of the pts with T1 BRCa, BMM were observed in 9.1% (19/208) pts versus 11.3% (7/62) in pts with T2 BRCa (p = 0.6). In pts with ER/PR-negative (-ve) BRCa, BMM were found in 7.7% (2/26) pts versus 9.9% (24/242) in pts with ER/PR-positive (+ve) BRCa (p = 0.27). BMM were detected in 12.3% (9/73) pts with Her-2/neu +ve BRCa and in 8.6% (16/187) pts with Her-2/neu -ve BRCa (p = 0.11). After completion of adjuvant therapy all pts with BMM (n = 26) converted to BM negative status. We conclude that BM status did not correlate with SLN status and occurs independently of lymphatic metastasis possibly through a different mechanism. BMM occur in node-negative pts and may assist in identifying pts at high risk for disease recurrence. Obtaining bone marrow aspirate from two locations resulted in a significant increase in detection of micrometastases.

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow Neoplasms; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Chemotherapy, Adjuvant; Female; Humans; Keratins; Lymphatic Metastasis; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Retrospective Studies; Sentinel Lymph Node Biopsy

The outcome of prostate cancer is highly unpredictable. To assess the dynamics of systemic disease and to identify patients at high risk for early relapse we followed the fate of disseminated tumor cells in bone marrow for up to 10 years and genetically analyzed such cells isolated at various stages of disease.. Nine hundred bone marrow aspirates from 384 patients were stained using the monoclonal antibody A45-B/B3 directed against cytokeratins 8, 18, and 19. Log-rank statistics and Cox regression analysis were applied to determine the prognostic impact of positive cells detected before surgery (244 patients) and postoperatively (214 patients). Samples from primary tumors (n = 55) and single disseminated tumor cells (n = 100) were analyzed by comparative genomic hybridization.. Detection of cytokeratin-positive cells before surgery was the strongest independent risk factor for metastasis within 48 months (P < .001; relative risk [RR], 5.5; 95% CI, 2.4 to 12.9). In contrast, cytokeratin-positive cells detected 6 months to 10 years after radical prostatectomy were consistently present in bone marrow with a prevalence of approximately 20% but had no influence on disease outcome. Characteristic genotypes of cytokeratin-positive cells were selected at manifestation of metastasis.. Cytokeratin-positive cells in the bone marrow of prostate cancer patients are only prognostically relevant when detected before surgery. Because we could not identify significant genetic differences between pre- and postoperatively isolated tumor cells before manifestation of metastasis, we postulate the existence of perioperative stimuli that activate disseminated tumor cells. Patients with cytokeratin-positive cells in bone marrow before surgery may therefore benefit from adjuvant therapies.

Topics: Bone Marrow Neoplasms; Comparative Genomic Hybridization; Humans; Kaplan-Meier Estimate; Keratins; Male; Neoplasm Metastasis; Prognosis; Prostatectomy; Prostatic Neoplasms; Risk Factors; Time

The role of circulating tumor cells (CTCs) in blood of primary breast cancer patients is still under investigation. We evaluated the incidence of CTCs in blood, we evaluated the correlation between CTCs and disseminated tumor cells (DTCs) in the bone marrow (BM), and we characterized CTCs for the expression of HER2, the estrogen receptor (ER) and the progesterone receptor (PR).. Blood of 431 patients with primary breast cancer were analyzed for EpCAM, MUC1 and HER2 transcripts with the AdnaTest BreastCancer (AdnaGen AG, Germany). Expression of the ER and PR was assessed in an additional RT-PCR. BM aspirates from 414 patients were analyzed for DTCs by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.. DTCs were found in 107/414 patients (24%), CTCs were detected in 58/431 (13%) patients. DTCs were associated with PR status of the primary tumor (P = 0.04) and CTCs significantly correlated with nodal status (P = 0.04), ER (P = 0.05), and PR (P = 0.01). DTCs in the BM weakly correlated with CTCs (P = 0.05) in blood. Interestingly, the spread of CTCs was mostly found in triple-negative tumors (P = 0.01) and CTCs in general were mostly found to be triple-negative regardless of the ER, PR and HER2 status of the primary tumor.. (1) Due to the weak concordance between CTCs and DTCs the clinical relevance may be different. (2) The biology of the primary tumor seems to direct the spread of CTCs. (3) Since the expression profile between CTCs and the primary tumor differs, the consequence for the selection of adjuvant treatment has to be evaluated.

Topics: Adenocarcinoma; Antigens, Neoplasm; Biomarkers, Tumor; Bone Marrow; Bone Marrow Neoplasms; Breast Neoplasms; Cell Adhesion Molecules; Chemotherapy, Adjuvant; Epithelial Cell Adhesion Molecule; Estrogens; Female; Humans; Keratins; Mucin-1; Neoplasm Proteins; Neoplasm, Residual; Neoplasms, Hormone-Dependent; Neoplastic Cells, Circulating; Neoplastic Stem Cells; Progesterone; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction

The sentinel lymph node (SLN) status has proven to accurately reflect the remaining axillary lymph nodes and represents the most important prognostic factor. It is unknown whether an association exists between the SLN status and the presence of bone marrow (BM) micrometastases. The objective of the present investigation was to evaluate whether or not such an association exists.. In the present investigation 410 patients with early stage breast cancer (pT1 and pT2

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Bone Marrow Neoplasms; Breast Neoplasms; Coloring Agents; Female; Humans; Keratins; Keratins, Type I; Keratins, Type II; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Prognosis; Prospective Studies; Sentinel Lymph Node Biopsy; Switzerland

Bone marrow (BM) is a prognostically relevant indicator organ of micrometastasis; however, the clinical importance of BM micrometastasis in gastric cancer patients is not yet known. In the present study, the BM of 267 consecutive patients with primary gastric cancer was examined for tumor cells using immunocytochemical techniques. Real-time quantitative RT-PCR was used to ensure that the tumor cells were detected properly. Among the 267 cases analyzed, 30 cases (11.2%) had cytokeratin-positive cells in the bone marrow. Positive findings were related to the tumor stage (P<0.05) and to the prominent depth of invasion (P<0.05). All patients with liver metastasis at operation had cytokeratin-positive cells in the BM. Recurrence of the disease was confirmed in 50 cases (18.7%); 4 of 30 (13.3%) in the cytokeratin-positive group and 46 of 237 (19.4%) in the cytokeratin-negative group. There were no significant differences in the 5-year survival rates between the cytokeratin-positive and cytokeratin-negative groups. Our study shows that BM micrometastasis increases according to tumor progression; however, only a subset of cancer cells may survive in the BM and finally evolve to a clinically apparent disease. Therefore it does not accurately predict the prognosis or recurrence of the disease.

Topics: Aged; Bone Marrow Cells; Bone Marrow Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Neoplasm Invasiveness; Neoplastic Cells, Circulating; Neovascularization, Pathologic; Prognosis; Stomach Neoplasms; Survival Rate

Comparative study of cytokeratin-positive (CK+) cells, isolated from bone marrow and lymph node micrometastases in patients with lung and esophageal cancer, was performed using the methods of immunocytochemistry and immunomagnetic separation. It was found that immunocytochemical analysis permitted the detection of CK+ cells in the smears of bone marrow of oncologic patients. All the samples of bone marrow with immunocytochemically demonstrated CK+ cells, contained also the cellular complexes marked by magnetic spheres. The method of positive immunomagnetic separation has some advantages for the intraoperative diagnosis. However, when either method was applied, among CK+ cells in bone marrow and lymph nodes some false-positive, questionable cellular forms and hemopoietic cells expressing epithelial markers were found. Theses results suggest that for verification of micrometastases in these organs, an additional study using the standard staining of the slides, is required.

Topics: Bone Marrow Cells; Bone Marrow Neoplasms; Humans; Immunohistochemistry; Immunomagnetic Separation; Keratins; Lymph Nodes; Lymphatic Metastasis

This prospective study is the first immunocytochemical investigation of the frequency and prognostic value of CK+ tumour cells in the bone marrow of patients with transitional cell carcinoma (TCC).. Bone marrow aspirates from 228 TCC patients were taken preoperatively. Cytospins were made and stained by immunocytochemistry using the monoclonal antibodies CK2 and A45-B/B3. 27 patients with no evidence of any malignant disease served as control group.. CK+ tumour cells were detected in 28% (63/228) of the TCC patients. No CK+ cells (0/27) were detected in the control group. In multivariate analysis the detection of > or =3 CK+ cells in bone marrow was an independent prognostic factor (hazard ratio=2.7, p<0.05) in patients with T2-4 tumour classification.. Disseminated CK+ cells play a role in the biology of tumour spread of TCC, and their immunocytochemical detection can be useful in assessing the prognosis of TCC patients with an invasive tumour.

Topics: Adult; Aged; Aged, 80 and over; Antibodies; Bone Marrow Examination; Bone Marrow Neoplasms; Carcinoma, Transitional Cell; Case-Control Studies; Female; Humans; Immunohistochemistry; Keratins; Kidney Neoplasms; Male; Middle Aged; Prognosis; Prospective Studies; Urinary Bladder Neoplasms

The presence of cytokeratin-positive cells in the bone marrow of breast cancer patients has been proven to be an independent prognostic factor. Their fate in primary breast cancer patients undergoing adjuvant therapy is of particular interest. We investigated the bone marrow status of 112 patients undergoing postoperative adjuvant treatment before and after therapy. A total of 373 patients with histologically confirmed primary breast cancer underwent bone marrow aspiration at the time of primary surgery. All patients were informed of their bone marrow status and offered repeat aspiration after 12 months. All patients were then treated with adjuvant chemotherapy, endocrine therapy or both based on current treatment recommendations. About 112 patients returned for a second bone marrow aspiration after a mean interval of 12 months following the initiation of adjuvant treatment. In 93 of 112 patients (83%) disseminated tumor cells had been found in the bone marrow before initiation of systemic chemo/endocrine therapy. At the time of follow-up sampling, after surgery and completion of adjuvant chemotherapy, the positivity rate dropped to 24%. Positive bone marrow status during follow-up was only associated with grading (p=0.020). Adjuvant treatment regimens are not able to completely eliminate cytokeratin-positive cells from the bone marrow. Prospective studies need to evaluate, whether these cells could become targets for additional adjuvant therapy.

Topics: Adult; Aged; Biomarkers, Tumor; Bone Marrow Neoplasms; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Chemotherapy, Adjuvant; Female; Follow-Up Studies; Humans; Keratins; Middle Aged; Neoplasm, Residual; Prognosis; Treatment Outcome

The prognostic relevance of disseminated cytokeratin-positive (CK+) tumor cells in the bone marrow of patients with different types of carcinoma has been demonstrated in several studies. In this prospective study, the frequency and prognostic value of CK+ tumor cells was investigated in the bone marrow of 55 consecutive patients with metastatic renal cell carcinoma (M1 RCC) in comparison with 256 M0 RCC patients from a previous study.. Aspiration of bone marrow from the anterior iliac crest was performed immediately before tumor resection in RCC patients. Cytospins were made and stained by immunocytochemistry using the APAAP (alkaline phosphatase-antialkaline phosphatase) protocol and monoclonal antibodies CK2 and A45-B/B3. Twenty-seven patients with no evidence of any malignant disease served as a control group.. CK+ tumor cells were detected in 42% (23 of 55 patients) of the M1 patients and 25% (63 of 256 patients) of the M0 patients (P <.01). No CK+ cells (0 of 27 patients) were detected in the control group. In the M1 group, CK- patients demonstrated a trend toward a better outcome compared with CK+ patients (log-rank test, P = .19). This difference was significant when applying a higher threshold (0-2 CK+ cells vs. > or = 3 CK+ cells; P <.05). On multivariate analysis, the detection of > or = 3 CK+ cells in the bone marrow was found to be an independent prognostic factor (P <.001).. The results of the current study indicate that disseminated CK+ cells play a role in the biology of tumor spread of RCC, and that their immunocytochemical detection can be useful in assessing the prognosis of patients with M1 disease.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Marrow Neoplasms; Carcinoma, Renal Cell; Case-Control Studies; Female; Humans; Immunohistochemistry; Keratins; Kidney Neoplasms; Male; Middle Aged; Neoplastic Cells, Circulating; Prognosis; Survival Analysis

The presence of disseminated tumor cells (DTC) in the bone marrow of breast cancer patients is an acknowledged independent prognostic factor. The biological metastatic potential of these cells has not yet been shown. The presence of putative breast cancer stem cells is shown both in primary tumors and distant metastases. These cells with a CD44(+)CD24(-/low) phenotype represent a minor population in primary breast cancer and are associated with self-renewal and tumorigenic potential. Recognizing the potential effect of prevalence of putative stem cells among DTC, we evaluated the bone marrow DTC.. We employed the double/triple-staining immunohistochemistry protocol and modified the established bone marrow cytokeratin (CK) staining protocol by adding steps for additional antigens, CD44 and/or CD24. We evaluated 50 bone marrow specimens, previously categorized as CK(+) from early breast cancer patients. CK(+) cells were examined for CD44 and CD24 expression by light microscopy, fluorescence microscopy, and spectral imaging.. We detected the putative stem cell-like phenotype in all CK(+) specimens. The mean prevalence of putative stem/progenitor cells was 72% and median prevalence was 65% (range, 33-100%) among the overall DTC per patient, compared with primary tumors where this phenotype is reported in <10% of cells.. This is the first evidence of the existence of the putative stem-like phenotype within the DTC in bone marrow in early breast cancer patients. All patients had a putative stem cell phenotype among the DTC and most individual DTC showed such phenotype. Future molecular characterization of these cells is warranted.

Topics: Bone Marrow; Bone Marrow Examination; Bone Marrow Neoplasms; Breast Neoplasms; CD24 Antigen; Female; Humans; Hyaluronan Receptors; Immunohistochemistry; Keratins; Neoplastic Stem Cells; Phenotype

We have previously developed a quantitative PCR (QPCR) technique for the detection of cytokeratin 19 (CK19) transcripts in blood and bone marrow and compared this to immunocytochemistry (ICC). Together, both have shown promise for monitoring therapeutic efficacy in patients with metastatic breast cancer. The aim of this study was to determine the feasibility and value of these assays for minimal residual disease (MRD) in monitoring efficacy of adjuvant therapy following surgery for primary breast cancer. Bone marrow aspirates and peripheral blood samples were taken at the time of surgery from patients with primary breast cancer and no evidence of metastases on conventional scans. These were tested for the presence of CK19 mRNA transcripts and cytokeratin positive cells. Follow-up bone marrow aspirates were taken at 3, 6, 12, 24, 36 and 48 months. Prior to surgery, 51% of patients displayed evidence of disseminated cancer cells in the bone marrow by either or both QPCR and ICC. Of 91 patients who had repeat samples assayed, 87% and 65% had positive results at some time using QPCR and ICC, respectively. All patients received adjuvant systemic therapy and in 44 cases where there was a positive result in either the pretreatment or 3-month aspirate, 32/44 (73%) showed a fall in CK19:ABL ratio (QPCR) and 15/24 (63%) showed a reduction in the number of cytokeratin-positive cells (ICC) during follow-up. These results indicate that MRD persists despite adjuvant therapy in a majority of patients with primary breast cancer up to 4 years following surgery.

Topics: Adult; Aged; Biomarkers, Tumor; Bone Marrow; Bone Marrow Neoplasms; Breast Neoplasms; Chemotherapy, Adjuvant; Feasibility Studies; Female; Follow-Up Studies; Humans; Immunohistochemistry; Keratins; Middle Aged; Neoplasm, Residual; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Treatment Outcome

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.

Topics: Aged; Bone Marrow Neoplasms; Breast Neoplasms; Case-Control Studies; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Growth Substances; Humans; Interleukin-6; Keratins; Middle Aged; Neoplasm Metastasis; Neovascularization, Pathologic; Prognosis; Regression Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity

The detection of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has proved prognostic significance in all stages of the disease. Further characterisation of those cells could help to improve the biological understanding of metastases, develop targeted therapies and define surface markers for enrichment techniques. The Thomsen-Friedenreich (TF) antigen has been shown to be a tumor specific antigen in breast cancer. The aim of this study was to investigate the expression of TF on DTC-BM in 25 patients. Bone marrow samples were first double-stained by a Cy3 conjugated cytokeratin (CK) antibody (ab) A45 B/B3 (IgG) and anti-TF ab Nemod 2 (IgM), followed by Cy2 conjugated goat anti-mouse IgM ab. For further characterisation samples were also double-stained with anti-TF ab Nemod 2 (IgM), followed by Cy2 conjugated goat anti-mouse IgM ab, and anti MUC1 ab A76-A/C7 IgG, followed by Cy3 conjugated goat anti-mouse IgG. CK positive DTC-BM showed co-expression of TF antigen in 22/23 patients (96%) and 61 of 62 detected cells (98%). Mononuclear BM cells without CK expression were also negative for TF. All of the TF positive cells showed strong MUC1 expression. This is the first study showing co-expression of CK and TF as markers of DTC-BM. Double staining experiments of TF and MUC1 expression showed that MUC1 is the carrier protein of TF in these cells. As TF is a specific marker of DTC-BM, it could be used as a target for antibody based therapy and immunomagnetic enrichment techniques for the isolation of DTC-BM.

Topics: Adult; Aged; Antigens, Tumor-Associated, Carbohydrate; Biopsy, Needle; Bone Marrow; Bone Marrow Examination; Bone Marrow Neoplasms; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Immunohistochemistry; Keratins; Microscopy, Fluorescence; Middle Aged; Mucin-1; Neoplasm Staging

Metastasis-the spread of tumour cells from a primary lesion to distant organs-is the main cause of cancer-related death, and bone marrow (BM) is a frequent site for the settlement of disseminated tumour cells. Many BM samples harbour isolated tumour cells, whereas tumour cell clusters, as the potential precursors of solid distant metastases, are rarely detected after current enrichment procedures. We have analysed BM samples from 43 patients with carcinomas of the breast, colon and ovaries; 41 of these patients had no clinical signs of overt metastases (stage M0). Tumour cells in BM were enriched with immunomagnetic beads coupled to monoclonal antibodies against both EpCAM and HER2/neu. After enrichment, tumour cells were identified by immunostaining with the anti-cytokeratin antibody A45-B/B3. In total, 886 CK-positive cells were detected in 16 (35%) samples after immunomagnetic enrichment as compared to 34 cells in 9 (21%) samples using Ficoll density centrifugation previously used as the standard enrichment technique. Most remarkably, clusters of 2 to 10 CK-positive cells were found in 75% of CK-positive samples enriched by immunobeads, whereas no CK-positive cell clusters were detected after Ficoll enrichment. The method described offers an excellent tool for the enrichment of micrometastatic tumour cell clusters; these clusters may represent the initial stage of development from a single disseminated tumour cell towards an overt metastasis.

Topics: Bone Marrow Neoplasms; Breast Neoplasms; Cell Separation; Centrifugation, Density Gradient; Colonic Neoplasms; Female; Ficoll; Humans; Immunohistochemistry; Immunomagnetic Separation; Keratins; Male; Neoplasm Metastasis; Neoplasm Staging; Ovarian Neoplasms

The detection of isolated tumor cells in bone marrow by immunocytochemistry (ICC) has been reported to predict progression of early-stage breast cancer. The most common staining procedure uses bright-field ICC with cytokeratin (CK) antibodies to label isolated tumor cells. However, this method can result in false-positive staining events. We used multicolor immunofluorescence (IF) to develop a more specific assay for detecting isolated tumor cells in marrow samples from breast cancer patients.. We compared ICC and IF side by side for detection of cancer cells and false-positive staining events on bone marrow aspirates from breast cancer patients, bone marrow from healthy donors, and healthy donor blood spiked with cancer cells. The primary target for isolated tumor cell detection was CK for both methods. IF used an additional set of antibodies to label hematopoietic cells (HCs).. The detection rate of CK+ events in breast cancer patient bone marrow aspirates was 18 (58%) of 31 for ICC and 21 (68%) of 31 for IF. However, with IF, 17 of 21 CK+ cases were stained with HC markers and thus were identified as false-positive events. A surprisingly high CK+ event rate was observed in healthy donor blood and marrow. In all healthy donor samples, CK+ events were readily identified as HCs by IF. Detection sensitivity of spiked cancer cells in donor blood was similar for both methods.. There is a high frequency of CK+ events in blood and marrow, and it is important to note that this is observed both in patients with and those without cancer. IF with multiple HC markers allows straightforward discrimination between CK+ cells of hematopoietic and nonhematopoietic origin.

Topics: Bone Marrow Examination; Bone Marrow Neoplasms; Breast Neoplasms; Female; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Keratins; Tumor Cells, Cultured

Interactions between the CXCR4 chemokine receptor in breast cancer cells and the ligand CXCL12/SDF-1alpha are thought to play an important role in breast cancer metastases. In this pilot study, CXCR4 expression along with other biomarkers including HER2-neu and EGFR, were measured in primary tumor samples of patients with operable breast cancer to test whether any of these biomarkers alone and in combination could indicate breast cancer with high likelihood of metastasizing to bone marrow. Cytokeratin (CK) positive cells in bone marrow were identified by flow-cytometry following enrichment with CK 7/8 antibody-coupled magnetic beads. Primary tumors (n = 18) were stained with specific antibodies for CXCR4, HER2-neu, EGFR, and PCNA using an indirect avidin-biotin horseradish peroxidase method. The majority of the patients had T2/T3 tumors (72%), or lymph node involvement (67%) as pathologic characteristics that were more indicative of high-risk breast cancer. High CXCR4 cytoplasmic expression was found in 7 of 18 patients (39%), whereas 6 of 18 patients (33%) were found to have CK positivity in bone marrow. The median number of CK(+) cells was 236 (range, 20-847) per 5 x 10(4) enriched BM cells. The presence of CK(+) cells in bone marrow was found to be associated with increased expression of CXCR4 alone or in addition to EGFR and/or HER2-neu expression (P = 0.013, P = 0.005, and P = 0.025, respectively) in primary tumors. Furthermore, three patients with high CK positivity (>236 CK(+) per 5 x 10(4) enriched bone marrow cells) in bone marrow exclusively expressed high levels of CXCR4 with EGFR/HER2-neu (P = 0.001). Our data suggest that high CXCR4 expression in breast cancer may be a potential marker in predicting isolated tumor cells in bone marrow. CXCR4 coexpression with EGFR/HER2-neu might further predict a particular subset of patients with high CK positivity in bone marrow.

Topics: Adult; Aged; Biomarkers, Tumor; Bone Marrow Neoplasms; Breast Neoplasms; Cytoplasm; ErbB Receptors; Female; Humans; Keratins; Middle Aged; Prognosis; Receptor, ErbB-2; Receptors, CXCR4

Detection of micrometastasis to the bone marrow can predict widespread disease and a poor prognosis of cancer patients. The purpose of this study was to evaluate the clinical significance of detecting micrometastasis in the bone marrow of esophageal cancer patients. Bone marrow and peripheral blood samples were obtained from 52 squamous esophageal cancer patients at the time of surgery. These samples were enriched by immunomagnetic separation and immunostained with an anti-cytokeratin antibody. Expression of vascular endothelial growth factor (VEGF) and cyclin D1 was examined in the primary tumors. Cytokeratin-positive cancer cells were observed in the bone marrow of 13 (25%) out of 52 patients. Among them, three patients also had cancer cells in the peripheral blood. The presence of bone marrow micrometastasis was correlated with lymph node metastasis (pN) but not associated with depth of tumors (pT). Detection of bone marrow micrometastasis was significantly correlated with VEGF expression of the primary tumors. Cumulative survival of patients with bone marrow micrometastasis was significantly lower than that of patients without it (p=0.0008). Hematogenous recurrence was more frequent in patients with bone marrow micrometastasis than in those without it (p=0.0004). Three patients who had local or dissemination recurrence did not have bone marrow micrometastasis. Detection of cancer cells in the bone marrow might be an indicator of early hematogenous metastasis in esophageal cancer patients. Intensive postoperative chemotherapy seems to be indicated for these patients.

Topics: Aged; Antigens, Neoplasm; Bone Marrow Neoplasms; Esophageal Neoplasms; Female; Humans; Immunomagnetic Separation; Keratin-19; Keratins; Male; Middle Aged; Serpins; Survival Analysis

Improving technologies for the detection and purification of bone marrow (BM) micrometastatic cells in breast cancer patients should lead to earlier prognosis of the risk of relapse and should make it possible to design more appropriate therapies. The technique used has to overcome the challenges resulting from the small number of target cells (one per million hematopoietic cells) and the heterogeneous expression of micrometastatic cell markers. In the present study, we have assessed the clinical relevance of current methods aimed at detecting rare disseminated carcinoma cells.. BM aspirates from 32 carcinoma patients were screened for the presence of micrometastatic cells positive for epithelial cell adhesion molecule and positive for cytokeratins, using optimized immunodetection methods. A comparison with data obtained for 46 control BM aspirates and a correlation with the clinical status of patients were performed.. We developed a sensitive and efficient immunomagnetic protocol for the enrichment of BM micrometastases. This method was used to divide 32 breast carcinoma patients into three categories according to their epithelial cell adhesion molecule status. These categories were highly correlated with the recently revised American Joint Committee on Cancer staging system for breast cancer, demonstrating the clinical relevance of this simple and reliable immunomagnetic technique. We also evaluated immunocytochemical detection of cytokeratin-positive cells and cytomorphological parameters. Immunocytochemistry-based methods for the detection of BM micrometastases did not provide any information about the clinical status of patients, but helped to refine the immunomagnetic data by confirming the presence of micrometastases in some cases. We also tested a new density gradient centrifugation system, able to enrich the tumor fraction of BM specimens by twofold to threefold as compared with standard Ficoll methods.. These improved methods for the detection of micrometastatic cells in patient BM should help clinicians to predict the clinical status of breast cancer patients at the time of surgery or treatment.

Topics: Adult; Antigens, Neoplasm; Bone Marrow Cells; Bone Marrow Neoplasms; Breast Neoplasms; Cell Adhesion Molecules; Epithelial Cell Adhesion Molecule; Humans; Immunohistochemistry; Immunomagnetic Separation; Keratins; Microscopy, Fluorescence; Middle Aged; Neoplasm Staging

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is a technique with the potential of improving the quantification of disseminated epithelial cells (DEC) in haematological tissues due to its exquisite sensitivity. This sensitivity may lead to false positivity. Immunocytochemistry (ICC) is regarded as the standard methodology to diagnose DEC. In this study, detection with ICC was compared with quantitative real-time RT-PCR for CK-19 and mammaglobin (hMAM) mRNA in bone marrow (BM) of patients with metastatic breast cancer (MBC). Bone marrow was aspirated from 14 control patients and from 29 patients with MBC. Mononuclear cells (MNC) were isolated. Immunostaining was carried out with the Epimet kit. Quantitative PCR was performed on the ABI Prism 7700. The CK-19 and hMAM mRNA quantities were normalised against beta-Actin and calculated relative to a calibrator sample (relative gene expression). All controls were negative by ICC and for hMAM expression measured by RT-PCR, whereas the median RGE value for CK-19 was 0.57. For the MBC patients, the median RGE for hMAM was 0 and 10 out of 25 (40%) tested positive. Median RGE for CK-19 was 2.9 and 20 out of 25 (80%) tested positive. With ICC, the median value was 1 stained cell per sample, and 15 out of 24 (62%) samples were positive. A correlation was observed between CK-19 and hMAM expression (r=0.7; P=0.0003), and between hMAM expression and ICC (r=0.6; P=0.003). CK-19 expression and ICC (r=0.9; P<0.0001) showed the strongest correlation. Reverse transcriptase-polymerase chain reaction for CK-19 resulted in a higher number of positive BM samples of patients with MBC than ICC. Since an excellent correlation is observed between ICC and RT-PCR, and RT-PCR is probably more sensitive with the advantage of being less observer dependent and thus also more easy to automate, we consider our quantitative real-time RT-PCR method as validated for the detection of DEC in the bone marrow of breast cancer patients.

Topics: Biomarkers, Tumor; Bone Marrow Examination; Bone Marrow Neoplasms; Breast Neoplasms; Epithelial Cells; Female; Humans; Immunohistochemistry; Keratins; Mammaglobin A; Molecular Diagnostic Techniques; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Uteroglobin

It is know that bone marrow micrometastases are an indicator of poor prognosis in patients with epithelial tumors. At the same time little is known if they reflect minimal residual disease or cells distribution and marked metastatic potential. We aimed to perform a clinical study, which though with little gathered clinical material has a direction--investigation of bone marrow in patients with colorectal cancer by the time of surgery, carefully rendering an account of staging and performing curative resection. In intraoperatively done bone marrow biopsy with following investigation for cytoceratin 19 positive cells we found ol presence of metastatic cells in some the studied patients. Finding micrometastases in patients with colorectal cancer is a marker for more agressive course of the disease and for the need to change some of recent methods of treatment.

Topics: Biomarkers, Tumor; Biopsy, Fine-Needle; Bone Marrow Examination; Bone Marrow Neoplasms; Colorectal Neoplasms; Humans; Keratins; Neoplasm Metastasis; Neoplasm, Residual

The presence of isolated tumor cells (ITC) in the bone marrow at the time of primary diagnosis indicates an increased risk for subsequent development of distant metastases in various solid tumors. This study evaluates the prevalence and prognostic significance of ITC in patients with primary carcinoma of the cervix uteri.. We immunocytochemically analyzed bone marrow aspirates of 130 patients with newly diagnosed carcinoma of the cervix uteri for the presence of cytokeratin(CK)-positive cells from May 1994 to January 2001. We used a quantitative immunoassay with the monoclonal anti-CK antibody A45-B/B3 and evaluated 2 x 10(6) bone marrow cells per patient. Patients were followed prospectively for a median of 43 (range, 1-85) months.. Isolated tumor cells were found in the bone marrow of 38 patients (29%). The presence of ITC did not correlate with the International Federation of Gynecology and Obstetrics (FIGO) tumor stage (P = 0.61), pelvic and paraaortal lymph node involvement (P = 0.41), histopathologic grading (P = 0.67), the histologic type of the carcinoma (P = 0.93), invasion of lymph nodes (P = 0.93) and blood vessels (P = 0.92), or with menopausal status (P = 0.17). The bone marrow status at the time of primary diagnosis did not correlate with the overall survival as estimated by Kaplan-Meier analysis (P = 0.30). However, distant metastases occurred in 5% of the patients (n = 5) with negative bone marrow status and in 15% of the patients (n = 6) with positive bone marrow status (P = 0.054). The median distant disease-free survival period was 78 months (95% confidence interval 73-82) in patients with negative bone marrow status and 72 months (95% CI 61-82) in patients with positive bone marrow status (P = 0.051). Multivariate analysis revealed the presence of ITC as a significant, independent risk factor for the subsequent development of distant metastases (relative risk 3.6, P = 0.046).. Despite the locoregional predominance of cervical carcinoma at the time of primary diagnosis, the presence of ITC in the bone marrow indicates an increased risk for the development of distant metastases. This information may prove useful to stratify patients for systemic treatment.

Topics: Adult; Bone Marrow Neoplasms; Carcinoma; Combined Modality Therapy; Female; Humans; Immunoenzyme Techniques; Keratins; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Prognosis; Proportional Hazards Models; Risk Factors; Survival Analysis; Uterine Cervical Neoplasms

Micrometastasis to the bone marrow can predict widespread disease and a poor prognosis of cancer patients after surgery. The purpose of this study was to evaluate the clinical significance of detecting micrometastasis in the bone marrow of gastric cancer patients.. Bone marrow and peripheral blood samples were obtained from 53 gastric cancer patients at the time of surgery. These samples were enriched by immunomagnetic separation and immunostained with an anti-cytokeratin antibody. Expression of vascular endothelial growth factor and erbB-2/HER2 was examined in the primary tumors.. Cytokeratin-positive cancer cells were observed in the bone marrow of 16 (30%) of 53 patients. Among them, two patients also had cancer cells in the peripheral blood. The presence of bone marrow micrometastasis was correlated with the depth of invasion and lymph node metastasis but was not associated with peritoneal dissemination. Detection of bone marrow micrometastasis was not correlated with vascular endothelial growth factor or HER2 expression in the primary tumors. Four patients with micrometastasis had recurrence in the liver or lungs, but this did not occur in patients without micrometastasis.. Detection of cancer cells in the bone marrow might be an indicator of postoperative hematogenous metastasis in gastric cancer patients.

Topics: Adult; Aged; Bone Marrow Neoplasms; Chi-Square Distribution; Endothelial Growth Factors; Female; Humans; Immunoenzyme Techniques; Immunomagnetic Separation; Intercellular Signaling Peptides and Proteins; Keratins; Lymphatic Metastasis; Lymphokines; Male; Middle Aged; Neoplasm Invasiveness; Neoplastic Cells, Circulating; Prognosis; Receptor, ErbB-2; Stomach Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Previous investigations have demonstrated the prognostic value of disseminated cytokeratin positive cells in bone marrow of patients with breast, gastric, colon and prostate cancer. We evaluated the potential of an immunocytochemical assay, using a monoclonal antibody against cytokeratin 18 (CK 18), for the detection of disseminated tumor cells in bone marrow aspirates of patients with transitional cell carcinoma.. Bone marrow aspiration was performed preoperatively on 128 patients with transitional cell carcinoma of various stages and on 27 controls with nonmalignant disease. Cytospin preparations of mononuclear bone marrow cells were incubated with a monoclonal anti-CK 18 antibody and stained using the alkaline phosphatase anti-alkaline phosphatase technique.. Of the patients with transitional cell carcinoma 29.7% and none of the controls had a CK 18 positive bone marrow result. A significant correlation between the incidence of CK 18 positive cells in bone marrow and invasive transitional cell carcinoma (p <0.01), lymph node involvement (p <0.01), medium/high grade transitional cell carcinoma (p <0.01) and tumor progression in recurrent transitional cell carcinoma (p <0.05) was demonstrated. Furthermore, the mean number of CK 18 positive cells in bone marrow aspirates of patients with stage M+ and/or N+ disease was nearly 3 times as high as that of patients without clinically evident metastatic disease (10.4 versus 3.8 CK 18 positive cells per patient).. A significant correlation between the incidence of CK 18 positive bone marrow results in patients with transitional cell carcinoma and established risk factors could be demonstrated in our study. Further prospective followup studies should be performed to determine the prognostic value of these findings.

Topics: Aged; Biomarkers, Tumor; Biopsy, Needle; Bone Marrow; Bone Marrow Neoplasms; Carcinoma, Transitional Cell; Disease Progression; Female; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Neoplasm Staging; Prognosis; Risk Factors; Survival Rate; Urinary Bladder Neoplasms

The presence of occult micrometastases in bone marrow (BM) of patients with early breast cancer increases the risk of relapse. Detection of circulation tumor cells in peripheral blood (PB) may also influence the patient's prognosis. Few data are available on the correlation between tumor cell dissemination in BM and PB in solid epithelial tumors. Twenty-milliliter blood samples were collected from PB of 42 patients with advanced breast cancer and centrifuged using the density gradient OncoQuick (OncoQuick Greiner BioOne, Frickenhausen, Germany). The BM aspirates available from 11 of the 42 patients were centrifuged using density centrifugation Ficoll. Tumor cell detection was performed by microscopy after cytospin preparation and immunocytochemical staining with the monoclonal antibody A45-B/B3. Cytokeratin-positive cells were detected in 23 patients (55%) in the PB and in three patients (27%) in the BM. A cohort with bone lesions as the only metastatic side showed a correlation as follows: 7 of the 11 patients (64%) had negative findings in BM and PB, whereas cytokeratin-positive cells in PB were present in 3 of these 11 patients (27%). The presence of visceral metastases was associated with the detection of cytokeratin-positive cells in the PB in 20 of the 31 patients (65%) in this subgroup. The density gradient OncoQuick in combination with immunocytochemical staining allows the detection of cytokeratin-positive cells in PB of patients with advanced breast cancer. The immunocytochemical detection of cytokeratin-positive cells in PB seems to be associated with the site of metastatic manifestation.

Topics: Bone Marrow; Bone Marrow Cells; Bone Marrow Neoplasms; Breast Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Neoplasm Metastasis; Neoplastic Cells, Circulating; Postmenopause; Prognosis; Treatment Outcome

Breast cancers in the early phase frequently undergo distant metastasis and survival of patients is greatly dependent on distant metastasis. The occurrence of micrometastasis has been suggested to relate with prognostic features of breast cancer, such as lymph node metastasis and the presence of vascular invasion. The aim of this study was to examine the presence of keratin-19 mRNA of epithelial tumors in bone marrow aspirates obtained from breast cancer patients and its possible correlation with tumor staging and disease-free survival.. Bone marrow samples were obtained from 59 breast cancer patients at the time of surgery. We separated the mononuclear fraction from the samples and carried out nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of keratin-19 mRNA with two different pairs of primers. After operation, the patients were followed up at 3-month intervals. We studied the possible correlation of the detection of keratin-19 mRNA with tumor size, nodal involvement, stage and recurrence rate.. Bone marrow micrometastasis was detected by nested RT-PCR for keratin-19 mRNA in one of four patients with ductal carcinoma in situ (DCIS), 13 of 30 patients with T1, 11 of 20 patients with T2 and all four patients with T3 lesion. Recurrence was observed in seven cases and all of them were positive for micrometastasis in bone marrow.. The method of nested RT-PCR to detect the presence of keratin-19 mRNA in bone marrow from patients with breast cancer is sensitive and reliable. Moreover, early recurrence was observed in the patients with the tumor mRNA detected in bone marrow. Additional studies with larger numbers of patients and longer follow-up are desirable.

Topics: Bone Marrow; Bone Marrow Neoplasms; Breast Neoplasms; Carcinoma, Ductal, Breast; Disease-Free Survival; Female; Humans; Keratins; Neoplasm Staging; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity

Several investigations indicate the prognostic value of disseminated cytokeratin positive tumor cells in bone marrow of patients with carcinoma of different origin. In this study we evaluated the prognostic significance of epithelial cells in bone marrow of patients with renal cell carcinoma (RCC).. Aspiration of bone marrow was performed preoperatively in 335 patients with RCC between 1990 and 1998. A total of 287 patients fulfilled all study inclusion (eg M0 R0 tumor stage) and exclusion (eg second malignancy during followup) criteria for the final analysis. Cytospin preparations were made after density gradient centrifugation of bone marrow samples and incubated with monoclonal antibodies directed against cytokeratin 18 (CK2) and pan-cytokeratin. Staining was performed using the alkaline phosphatase-anti-alkaline phosphatase method and 256 samples were evaluated. RESULTS In 25% (63 of 256) of the patients cytokeratin positive (CK+) cells were detected in bone marrow. Tumor progression (defined as tumor associated death, local recurrence or new metastases) was present in 12% (31 of 256) during the followup period (median 40 months), and 14% (9 of 63) with CK+ cells and 11% (22 of 193) with negative bone marrow status exhibited tumor progression. Survival analysis (log-rank test) showed no significant difference between the CK+ and cytokeratin negative group. The detection of CK+ cells was not an independent prognostic parameter in multivariate analysis (Cox regression model).. These results indicate that the immunocytochemical detection of disseminated cytokeratin positive tumor cells in the bone marrow of patients with RCC has no prognostic significance.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy, Needle; Bone Marrow; Bone Marrow Neoplasms; Carcinoma, Renal Cell; Cell Count; Disease Progression; Epithelial Cells; Female; Humans; Immunoenzyme Techniques; Keratins; Kidney Neoplasms; Male; Middle Aged; Neoplasm Staging; Regression Analysis; Survival Analysis

Bone marrow is a prognostically relevant indicator organ for micrometastasis. In the present study, real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect disseminated gastric cancer cells in bone marrow. We compared CEA, CK18 and CK20 expression using four gastric cancer cell lines and three normal tissue cell lines in order to select the most appropriate marker for detection of disseminated gastric cancer cell in bone marrow. CK20 proved to be the most promising marker since the expression level of normal cell lines was extremely low and about 50--100-fold differences were found between gastric carcinoma cell lines and normal tissue cell lines. We also screened bone-marrow RNA of 47 patients with gastric cancers, using this system. Among the three markers we tested, with only about CK20 could we find that 27 of 47 patients were positive. Though long-term clinical follow up studies are needed to evaluate the clinical significance of this method, real time quantitative RT-PCR is sensitive and quantitative for detection of micrometastasis in bone marrow.

Topics: Adenocarcinoma; Biomarkers, Tumor; Bone Marrow Neoplasms; DNA Primers; Female; Humans; Intermediate Filament Proteins; Keratin-20; Keratins; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Stomach Neoplasms

The presence of disseminated tumor cells in bone marrow is considered to be a premetastatic state, which is called micrometastasis. To evaluate the relationship between micrometastasis and cellular adhesion molecules in the primary lesion, E-cadherin and beta-catenin were immunohistochemically investigated. Methods. Fifty-eight patients with non-small cell lung cancer who underwent a complete resection were entered into this study. Tumor cells in bone marrow aspirates were detected by immunohistochemistry using cytokeratin (CK) 18. Immunohistochemical studies of E-cadherin and beta-catenin were performed in the corresponding primary tumor.. CK-positive cells were detected in the bone marrow aspirates from 27 of 58 patients. A reduced expression of the E-cadherin and beta-catenin was found in 16 (27.6%) and in 22 (37.9%) of 58 patients, respectively. In 26 cases with a reduced expression of E-cadherin and/or beta-catenin, 16 cases had CK-positive cells, whereas 11 of 32 cases with normal expression of both factors had CK-positive cells (P=.0392). The patients with micrometastasis demonstrated an earlier recurrence (P =.0642) and a significantly poorer survival (P =.0437) than those without such cells.. Micrometastasis in the bone marrow might be a significant predictor of poor prognosis, and a reduced expression of E-cadherin and beta-catenin are important determinants for the metastatic capability of individual cancer cells.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; beta Catenin; Bone Marrow Neoplasms; Cadherins; Carcinoma, Squamous Cell; Cytoskeletal Proteins; Disease-Free Survival; Female; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Male; Middle Aged; Predictive Value of Tests; Prognosis; Risk Factors; Survival Rate; Trans-Activators

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.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Bone Marrow Cells; Bone Marrow Neoplasms; Disease Progression; Female; Follow-Up Studies; Humans; Keratins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Predictive Value of Tests; Stomach Neoplasms; Survival Analysis; Treatment Outcome

To study the invasion-related molecule urokinase-type plasminogen activator receptor (u-PAR) expressed by disseminated tumor cells as a biologic predictor of poor survival in a large prospective series of patients with gastric cancer.. In 156 gastric cancer patients (prospective series), disseminated tumor cells in the bone marrow and the u-PAR expressed by these tumor cells were determined by cytokeratin (CK) 18 immunocytochemistry and u-PAR/CK18 double immunocytochemistry.. In contrast to the mere detection of disseminated tumor cells at primary surgery, the additional evidence of u-PAR on these cells correlated significantly with pathologic T stage (P =.0474) and the expression of u-PAR (P =.0093) and plasminogen-activator inhibitor 1 (P =.0145) in the primary tumor (immunohistochemistry, chi(2)). Kaplan-Meier analysis revealed no association with prognosis for the mere detection of disseminated tumor cells. In contrast, a significant association was seen between detection of u-PAR on these cells and shorter disease-free (P <.0001) and overall survival (P <.0001). Multivariate analysis revealed that u-PAR on disseminated tumor cells at the time of primary surgery is an independent prognostic factor for disease-free (95% confidence interval [CI], 1.72 to 3.21; P =.024) and overall survival (P =.0049; relative risk, 2.89; 95% CI, 1.92 to 4.30).. This is the first large study to show that u-PAR, detected on disseminated tumor cells in the bone marrow, is an independent prognostic parameter in gastric cancer, in contrast to the mere detection of minimal residual disease (MRD). u-PAR may be a promising marker to define a critical subpopulation of disseminated tumor cells and a target to eliminate MRD. Molecular phenotyping of MRD is critical for defining its individual clinical relevance.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Bone Marrow Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Neoplasm Staging; Neoplasm, Residual; Plasminogen Inactivators; Prognosis; Prospective Studies; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Stomach Neoplasms; Survival Analysis; Urokinase-Type Plasminogen Activator

The long-term prognosis after surgical therapy for esophageal carcinoma depends on tumor stage and completeness of resection. Similarly to other epithelial tumors, the presence of micro deposits of neoplastic cells in the bone marrow may indicate residual disease and the potential for recurrence. This study assesses the prevalence of bone marrow-disseminated tumor cells in patients undergoing surgical resection for esophageal carcinoma. In addition, we investigated the agreement between immunohistochemical and molecular techniques for the detection of micrometastases in a subgroup of patients.. Between January 1998 and November 1999, forty-eight patients with adenocarcinoma of the esophagogastric junction (n = 29) or squamous cell carcinoma of the thoracic esophagus (n = 19) and no evidence of overt metastatic disease entered the study. An immunohistochemical assay (capable of detecting 1 carcinoma cell in 7 x 10(5) bone marrow cells) was used to test bone marrow obtained by flushing a resected rib or by needle aspiration either of the iliac crest or of a rib. A polymerase chain reaction (PCR) molecular technique was also used to identify bone marrow and peripheral blood epithelial cells.. Cytokeratin-positive cells were found in 79.1% of the bone marrow samples obtained from the rib, and in only 8% of the needle aspirates either from the iliac crest or from a contiguous rib: This difference is probably explained by the improved removal of metastatic cells with the flushing of the rib. Comparable results were obtained at a qualitative level by the PCR technique on bone marrow. In addition, PCR-positive results were found in 3 of 18 peripheral blood samples. There was no association with tumor type, neoadjuvant therapy, or lymph node status. Patients with a pT3 or pT4 tumor showed, at a borderline statistical level, a higher proportion of cytokeratin-positive cells in the flushed rib.. Bone marrow-disseminated tumor cells are present in the resected rib of a high proportion of patients undergoing esophagectomy for carcinoma, and immunohistochemistry seems to be the method of choice for their quantitative assessment. However, the prognostic and therapeutic implications of this finding need further investigation.

Topics: Adenocarcinoma; Adult; Aged; Base Sequence; Bone Marrow Examination; Bone Marrow Neoplasms; Carcinoma, Squamous Cell; Cardia; DNA Primers; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Polymerase Chain Reaction; Prognosis; Stomach Neoplasms

Prostate cancer commonly metastasises to the bones. Detection of bone marrow micrometastases (BMM) may give important information that helps define treatment strategies. This study was undertaken to analyse BMM in early prostate cancer patients and to determine the accuracy of immunohistochemical (IHC) and morphological methods in detecting cancerous cells. Preoperative core bone marrow biopsy (BMB) was performed in 103 patients with T1-2, N0, M0 prostate cancer after neoadjuvant androgen blockade. BMB were examined by IHC using monoclonal antibodies for cytokeratins (CK) (18, 19, PAN) and by cytomorphology of IHC-positive cells. In 103 patients, BMM were detected in 2 cases (2%) and an additional 3 cases (3%) were classified as suspicious. IHC alone revealed positive cells in 19 patients (18%). Cytomorphology disclosed IHC false-positive staining of some apparently normal bone marrow elements such as plasmocytes. The study shows a rather low rate of BMM in early prostate cancer. It also stresses the importance of cytomorphology as an adjunct to IHC as IHC alone may not be sufficient and appropriate for BMM detection.

Topics: Adult; Aged; Biomarkers, Tumor; Biopsy; Bone Marrow Neoplasms; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Prospective Studies; Prostatectomy; Prostatic Neoplasms

This prospective study aimed to evaluate the detection of micrometastases in bone marrow of patients with suspected pancreatic and ampullary cancer and to determine their predictive value on overall survival.. Between December 1997 and December 1998, 35 patients (19 male, 42-77 years) with suspected pancreatic and ampullary cancer underwent diagnostic laparoscopy as a final staging procedure before exploration. Bone marrow was aspirated from the iliac crest at the beginning of laparoscopy. Mononuclear cells were isolated and stained using the specific monoclonal antibody CAM 5.2.. Cytokeratin-positive cells were detected in 12/35 (34%) of all patients. In the 31 patients with a final diagnosis of carcinoma, a positive staining was found in 10/31 (32%) of the bone marrow aspirates. After a median follow-up of 17 months (2-24), 15/31 (48%) patients had died: 7/10 (70%) with and 8/21 (38%) without micrometastases (* P<0.04). All four patients who turned out to have chronic pancreatitis were alive without malignancy. In two of these four patients, distinct cytokeratin-positive cells were seen.. Micrometastases in bone marrow of patients with the final diagnosis pancreatic or ampullary carcinoma seem to predict a significantly shorter survival. However, clinical use of cytokeratin markers cannot be recommended at present, because false-positive staining was found.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers; Biomarkers, Tumor; Bone Marrow Cells; Bone Marrow Neoplasms; Common Bile Duct Neoplasms; Female; Humans; Immunohistochemistry; Inhalation; Keratins; Laparoscopy; Male; Middle Aged; Neoplasm Staging; Pancreatic Neoplasms; Prospective Studies; Survival Analysis

The search continues to find methods to more effectively distinguish colorectal carcinoma patients who could be separated into high-risk and low-risk categories. Investigators have reported on the detection of occult micrometastases in bone marrow using antibodies to cytokeratin, which is a marker of epithelial cells but which has no tissue specificity, as opposed to villin, a cytoskeletal protein that is specifically involved in the formation of brush-border microvilli in the small intestine and colon epithelium. Specificity and sensitivity of antibody to villin (ID2C3) and antibody to cytokeratin (A45-B/B3) were first studied in normal bone marrow and in a test system in which cancer cell lines were mixed in normal bone marrow. In a preliminary study including 16 colorectal carcinoma patients, we compared the number of villin-positive cells with cytokeratin-presenting cells. As A45-B/B3, ID2C3 was determined to be sensitive enough to detect one cancer cell in 10(6) hematopoietic cells. Staining of hematopoietic cells with irrelevant antibody and a light staining of megakaryocytes with ID2C3 limited the specificity of the method. In colorectal carcinoma patients, correlation between ID2C3 and A45-B/B3 was 94%. Sensitivity and specificity of ID2C3 antibody to villin were satisfactory. Its clinical relevance must be investigated in further studies.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal; Biomarkers, Tumor; Bone Marrow Cells; Bone Marrow Neoplasms; Carcinoma; Carrier Proteins; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Microfilament Proteins; Middle Aged; Sensitivity and Specificity; Tumor Cells, Cultured

An autopsy case of primary hepatic neuroendocrine carcinoma is described. A 72-year-old man had a large tumor mass measuring 22 cm in its greatest diameter and localized to the right, left and caudal lobes of the non-cirrhotic liver. Microscopically, the tumor was composed of middle-sized pleomorphic cells organized in ribbons or trabeculae, with scanty intersecting fibrous septae. Immunohistochemically, the tumor cells were positive for multikeratin C11, chromogranin A and synaptophysin. The patient also had metastases in the bone marrow. No alternative primary source of endocrine tumor was detected. The patient died 4 days after presentation.

Topics: Aged; Biomarkers, Tumor; Bone Marrow Neoplasms; Carcinoma, Neuroendocrine; Chromogranin A; Chromogranins; Fatal Outcome; Humans; Immunohistochemistry; Keratins; Liver Neoplasms; Male; Synaptophysin

We have compared three different RT-PCR procedures to measure cytokeratin 19 (CK19), carcinoembryonic antigen (CEA) and mucin MUC1 gene expression in order to determine their diagnostic value in detecting tumour cells in bone marrow aspirates of patients with operable breast cancer. In an experimental model, the best sensitivity was observed for CK19 and MUC1 RT-PCR assays, although only the CEA and CK19 assays showed good specificity. The study of 42 patients showed that a 'CK19 positive/CEA positive' RT-PCR assay in bone marrow correlated positively with a positive axillary lymph node status (N(0) versus N(1-3), P<0.05). Both assays were also positive in 17% of node negative patients. RT-PCR assays were more sensitive in bone marrow than in peripheral blood. Our results suggest that CK19 and CEA RT-PCR assays are powerful methods for detecting disseminated breast cancer cells. A larger study with long-term follow-up is required in order to clarify their clinical usefulness.

Topics: Biomarkers, Tumor; Bone Marrow Neoplasms; Breast Neoplasms; Carcinoembryonic Antigen; Female; Humans; Keratins; Lymphatic Metastasis; Mucin-1; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Tumor Cells, Cultured

We report a highly sensitive method to detect rare human breast cancer cells, which combines an immunomagnetic separation (IMS) using antibody BM2 against MUC-1 with cytokeratin-19 (CK19) and the reverse transcriptase/polymerase chain reaction (RT-PCR). The IMS-RT-PCR technique allows the detection of 1 tumor cell/10(7)-10(8) mononuclear cells. This is at least ten times more sensitive than CK19 RT-PCR alone, or immunocytochemistry. All 117 peripheral blood and 8 bone marrow samples obtained from healthy donors as negative controls were positive for beta2-microglobulin by RT-PCR but negative for CK19 by IMS-RT-PCR or RT-PCR alone. Out of 26 bone marrow samples from breast cancer patients, 18 had CK19 transcripts detectable by IMS-RT-PCR. In contrast, only 14 and 13 samples from the 26 were found to be positive by RT-PCR alone or by routine immunocytochemical staining. In conclusion, IMS-RT-PCR for CK19 is a highly sensitive and specific method for detecting very low numbers of micrometastatic breast cancer cells in bone marrow amidst an excess of nonmalignant cells. For the early diagnosis of disseminating disease, this assay is more efficient than RT-PCR alone and routine immunocytochemistry.

Topics: Bone Marrow; Bone Marrow Neoplasms; Breast Neoplasms; Cell Separation; Female; Humans; Immunomagnetic Separation; In Vitro Techniques; Keratins; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

Carcinoma that has metastasized to the central nervous system (CNS) poses a particular clinical problem regarding confirmation of the diagnosis and subsequent management. Prior to excision, thorough evaluation for coexisting systemic disease is essential, but current imaging techniques are limited by their spatial resolution and under-stage many patients. We evaluated the potential utility of bone-marrow evaluation for micrometastatic cells in patients with CNS metastasis. Bone-marrow aspirates were examined for cytokeratin-positive cells in 12 consecutive patients who presented with symptomatic space-occupying lesions of the CNS. These patients had previously undergone surgical excision of either gastrointestinal or breast cancers. All twelve had micrometastases in their bone marrow at the time of presentation with the CNS disease and all had a fatal outcome within 13 months. In nine of the 12 patients, bone-marrow micrometastases were the only evidence for systemic spread. Three patients had elevated serum tumour markers and two of these had radiologically detectable recurrence elsewhere. Bone-marrow micrometastases indicate concurrent systemic involvement and a poor prognosis. The results suggest that bone-marrow evaluation for systemic spread is a useful diagnostic adjunct and should be performed before considering diagnostic biopsy or excision.

Topics: Adenocarcinoma; Biomarkers, Tumor; Bone Marrow Examination; Bone Marrow Neoplasms; Brain; Brain Neoplasms; Breast Neoplasms; Carcinoma; Carcinoma, Ductal, Breast; Female; Flow Cytometry; Gastrointestinal Neoplasms; Humans; Immunohistochemistry; Keratins; Neoplasm Staging; Predictive Value of Tests; Prospective Studies; Tomography, X-Ray Computed

Amplification of cytokeratin 19 (CK19) transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) has been shown to be a highly sensitive assay for the detection of bone marrow micrometastases (BMM) of breast cancer, but recent studies have demonstrated the occurrence of false-positive results due to low-level, illegitimately transcribed CK19 in normal bone marrow tissue. One approach to solve this problem is to develop a quantitative CK19 RT-PCR assay and to introduce a cut-off value which can distinguish between illegitimate expression and cancer-specific expression levels. In the present paper, we describe a quantitative CK19 RT-PCR assay using a real-time automated PCR system. The number of CK19 transcripts was normalized to that of GAPDH transcripts as an internal control for quality and quantity of cDNA. The cut-off value for the ratio of CK19 to GAPDH transcripts was set at 10(-4) since the ratio never exceeded this value in the control bone marrow samples (n = 12). In total, 117 bone marrow aspirates from stage I - III patients with invasive breast cancers were subjected to CK19 RT-PCR assay and immunocytological examination. Forty (34.2%) were found to be BMM-positive by CK19 RT-PCR assay whereas only three (2.6%) were found to be BMM-positive by immunocytology. Multivariate analysis has shown that occult BMM detected by CK19 RT-PCR is a significant risk factor for relapse, being independent of axillary lymph node metastases.

Topics: Bone Marrow Neoplasms; Breast Neoplasms; Humans; Keratins; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity

Some patients with breast cancer currently undergo bone marrow biopsy to make clinical decisions regarding therapy; however, lobular carcinoma can be difficult to detect in routine histologic sections. The authors reviewed retrospectively all available bone marrow biopsies from patients with lobular carcinoma diagnosed between January, 1, 1989, and September, 25, 1997, at the City of Hope National Medical Center to identify useful morphologic features and to determine the utility of pan-keratin immunohistochemical (IHC) staining. A total of 65 biopsies from 54 patients were reviewed. Thirteen of the 65 biopsies were classified initially as containing metastatic tumor based on histologic features alone. With the addition of keratin IHC, seven additional cases of metastatic disease were detected. Forty of the 54 patients received stem cell replacement or autologous bone marrow transplantation. Disease-free survival after high-dose chemotherapy with stem cell replacement or autologous bone marrow transplantation was stratified into three groups based on hematoxylin and eosin (H&E) staining and IHC results. Two-year disease-free survival was 33% for the H&E-/IHC+ group versus 90% for the H&E-/IHC- group (p = 0.005) among patients clinically free of disease at the time of stem cell replacement or autologous bone marrow transplantation. Two-year disease-free survival was 0% in the H&E+/IHC+ group (p = 0.04, compared with the H&E-/ IHC+ group). The authors conclude that routine morphologic examination without the aid of keratin IHC is unreliable in detecting clinically relevant metastatic lobular carcinoma in bone marrow biopsies. These findings suggest that pan-keratin immunostaining may be indicated on bone marrow biopsy specimens from lobular carcinoma patients if the biopsy appears histologically negative for metastatic tumor on H&E sections.

Topics: Adult; Aged; Biopsy; Bone Marrow Neoplasms; Breast Neoplasms; Carcinoma, Lobular; Disease-Free Survival; Female; Follow-Up Studies; Humans; Immunohistochemistry; Keratins; Middle Aged; Regression Analysis; Retrospective Studies

Reverse transcriptase-polymerase chain reaction (RT-PCR) assay for prostate-specific antigen and immunocytochemistry for cytokeratin-18 (CK-18) are tests for the detection of microdisseminated carcinoma of the prostate. Bone marrow aspirates and peripheral venous blood from 50 patients with clinically organ-confined prostate cancer were examined. The rate of positive results was independent of the pT stage, serum PSA, and previous antiandrogen treatment. RT-PCR and immunocytochemistry have to be tested under standardized conditions in prospective trials, and the results have to be compared to the serum PSA follow-up.

Topics: Aged; Biopsy, Needle; Bone Marrow; Bone Marrow Neoplasms; Humans; Keratins; Male; Middle Aged; Neoplasm Staging; Neoplastic Cells, Circulating; Predictive Value of Tests; Prognosis; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction

Atypical epithelial cells in the bone marrow of patients with ovarian cancer were evaluated using immunohistochemical techniques. We investigated cytospin preparations of bone marrow taken from 9 women with benign ovarian tumors and 59 women with malignant ovarian tumors. Two monoclonal antibodies (NCL-C11 and NCL-CA 125) were used. With both antibodies we were able to detect keratin and CA 125 antigen expression in the bone marrow of 9 (18.4%) of the patients with ovarian cancer. With regard to the wide histological differentiation of ovarian carcinomas, the presence of atypical epithelial cells in the bone marrow was required as a prognostic factor for survival and relapses. This should be investigated in a larger study group.

Topics: Adult; Aged; Bone Marrow; Bone Marrow Neoplasms; CA-125 Antigen; Female; Humans; Keratins; Middle Aged; Ovarian Neoplasms; Prognosis; Survival Rate

After radical prostatectomy for clinically localized prostate cancer, biochemical progression is seen in up to 40% of the patients due to persistent local and/or systemic remnants. Isolated disseminated carcinoma cells, undetectable by current staging methods, are of special interest as potential precursors of subsequent overt metastases. In the present study, immunohistochemistry (IHC) was performed to evaluate simultaneously the frequency of occult carcinoma cells in both lymph nodes (LNs) and bone marrow (BM) obtained from the iliac crests of 45 prostate cancer patients with untreated stage T(1-3) pN0 M0 prostatic carcinoma. IHC using monoclonal antibodies (MAbs) against epithelial cytokeratins was performed on 521 paraffin-embedded LNs histopathologically classified as tumorfree (pN0), as well as on BM cytospin preparations. To confirm the prostatic origin of positive cells in LNs, additional IHCs for prostate-specific antigen (PSA) and epithelial glycoproteins were performed. In total, isolated tumor cells in LNs and/or BM were detected in 17 of the 45 patients. Parameters such as tumor stage, grade and volume of the primary tumor as well as blood serum PSA levels could not detect patients harboring disseminated single tumor cells in LNs or BM. Following a median observation time of 24.9 months, no significant correlation between IHC positivity and PSA progression as a measure of early relapse was observed. Although the overall incidence of occult tumor cell spread corresponds to similar incidence of relapses after radical prostatectomy as reported by others, the fate of these cells needs to be evaluated in longer follow-up studies.

Topics: Bone Marrow Neoplasms; Humans; Immunohistochemistry; Keratins; Lymphatic Metastasis; Male; Neoplasm Staging; Prostate-Specific Antigen; Prostatectomy; Prostatic Neoplasms

The work aimed to evaluate the sensitivity and specificity of the cytokeratin (CK) 19 reverse transcriptase/polymerase chain reaction (RT-PCR) for the detection of occult breast cancer in bone marrow and leukapheresis products.. Peripheral blood and bone marrow samples, obtained from 96 and 8 healthy donors respectively, served as negative controls. A total of 115 bone marrow samples and 29 leukapheresis samples from routine patients with breast cancer were analysed by CK19 RT-PCR. The PCR results were compared with those from routine immunocytology for CK8, 18, 19.. The CK19 RT-PCR technique with primer pairs from Datta et al. (J Clin Oncol 12: 475-482, 1994), using an annealing temperature of 72 degrees C, allowed the detection of one tumour cell in 10(7) mononuclear cells. None of the control samples (96 peripheral blood and 8 bone marrow) that were positive for beta2-microglobulin by RT-PCR showed a signal for CK19. However, expression of CK19 mRNA was observed in 40.87% (70/115) of bone marrow and in 24.13% (7/29) of leukapheresis samples of patients with breast cancer. Standard immunocytology and PCR were combined for the detection of tumour cells. Five of the 65 bone marrow samples were found to be positive by CK19 RT-PCR, but were negative with the immunocytology method.. RT-PCR using CK19-specific primers and optimal experimental conditions is a reliable and specific method for the detection of micrometastatic breast cancer cells.

Topics: Bone Marrow Neoplasms; Breast Neoplasms; DNA Primers; Female; Humans; Keratins; Leukapheresis; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

Minimal residual disease in patients with operable pancreatic carcinoma is frequently missed by current noninvasive tumour staging. We applied an immunocytochemical cytokeratin assay that allows identification of individual pancreatic carcinoma cells disseminated to bone marrow. Prior to therapy, bone marrow was aspirated from the upper iliac crest of 48 patients with ductal adenocarcinoma of the pancreas at various disease stages and an age-matched control group of 33 noncarcinoma patients. Tumor cells in cytologic bone marrow preparations were detected with monoclonal antibodies (mAbs) CK2, KL1, and A45-B/B3 to epithelial cytokeratins (CK) using the alkaline phosphatase antialkaline phosphatase method. CK-positive cells were found in 14 (48.4%) of 31 cancer patients treated with curative intent and in 10 (58.8%) of 18 patients with extended disease. The overall frequency of these cells was 1 to 83 per 5x10(5) mononuclear cells with no significant differences between patients at different tumor stages and lymph node involvement. After a median follow-up of 22.8 months (range 3-48 months), 6 (40.0%) of 15 patients who underwent complete surgical resection but had tumor cells in bone marrow presented with distant metastasis and 7 (46.7%) had local relapse compared to none of 12 corresponding patients without such cells (p<0.05). Univariate survival analyses revealed that the presence of CK-positive cells was predictive of reduced overall survival. In conclusion, anticytokeratin mAbs are reliable probes for the immunocytochemical detection of single pancreatic cancer cells disseminated to bone marrow. Thus the described technique may help identify patients with pancreatic cancer and at potentially high risk of early metastatic relapse. The results promise to be of important assistance for determining prognosis and the consequences in therapy of early stage pancreatic cancer.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Bone Marrow Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Pancreatic Neoplasms; Prognosis; Prospective Studies

The detection of cytokeratin-positive bone marrow cells has been considered a prognostic factor in numerous malignant tumors. We investigated whether this was also valid for localized prostate cancer. Bone marrow aspirates were taken prior to radical prostatectomy from 169 consecutive patients with pT1/2 pN0 G1-3 adenocarcinoma of the prostate. The immunocytochemical detection of cytokeratin no. 18 (CK 18)-positive cells using monoclonal antibody CK 2 was interpreted as micrometastasis. Repeat marrow aspirations were performed at 6 months postoperatively and once a year thereafter. The patients were re-examined over a period of at least 10 and a maximum of 72 months (median 32 months). An increase in prostate specific antigen >/=0.5 ng/ml was considered a biochemical "relapse". One hundred and fifty-four patients had evaluable bone marrow aspirates, of which 74.7% were CK 18-negative and 25.3% positive. The latency period for biochemical relapse was 1481 days (median) in the CK 18-negative group and 1106 days (median) in the CK 18-positive group. This difference was not statistically significant. The CK 18-positive aspirates (n = 39) showed one positive cell in 20 cases, two positive cells in 8 and three or more positive cells in 11 cases. The preoperative number of cells had no statistically significant effect upon the onset of biochemical relapse. Only patients with three or more CK 18-positive cells tended to have a poorer prognosis. One hundred and thirteen patients had evaluable bone marrow aspirates pre- and postoperatively. Postoperative persistence or occurrence of CK 18-positive cells did not affect the outcome of the disease. The detection of CK 18-positive cells in bone marrow does not influence the prognosis of patients with localized prostate cancer within a period of 32 months (median). Solely a subgroup of patients showing a large preoperative number of CK 18-positive cells seems to tend to an unfavorable course of the disease. Thus, further studies are necessary aiming at a more detailed characterization of these cells.

Topics: Adenocarcinoma; Aged; Bone Marrow Examination; Bone Marrow Neoplasms; Disease-Free Survival; Epithelial Cells; Humans; Keratins; Male; Middle Aged; Prognosis; Prostate-Specific Antigen; Prostatectomy; Prostatic Neoplasms; Recurrence

Dissemination of single tumor cells to the bone marrow is a common event in cancer. The clinical significance of cytokeratin-positive cells detected in the bone marrow of cancer patients is still a matter of debate. In gastric cancer, overexpression of the receptor (uPAR or CD87) for the serine protease urokinase-type plasminogen activator (uPA) in disseminated cancer cells indicates shorter survival of cancer patients. A new immunofluorescence approach, applying confocal laser scanning microscopy, is introduced to locate CD87 antigen in cytokeratin-positive tumor cells and to quantify the CD87 antigen by consecutive scanning. At first, cytokeratin 8/18/19-positive carcinoma cells are identified at excitation wavelength 488 nm using monoclonal antibody A45B/B3 to the cytokeratins and goat anti-mouse IgG labeled with the fluorochrome Alexa488. Next, CD87 in tumor cells is identified by chicken antibody HU277 to the uPA-receptor and goat anti-chicken IgY labeled with fluorochrome Alexa568 (excitation wavelength 568 nm) and the fluorescence signal quantified on a single cell basis using fluorescently labeled latex beads as the fluorescence reference. From 16 patients with gastric or esophageal carcinoma, bone marrow aspirates were obtained, stained for cytokeratins and CD87 and then subjected to laser scanning fluorescence microscopy. Three of six gastric cancer patients had tumor cells present in the bone marrow of which 2 stained for CD87. Three of ten esophageal carcinoma patients had tumor cells in the bone marrow, all three samples stained for CD87. CD87-positive tumor cells were also dissected from stained bone marrow aspirates by laser microdissection microscope to allow analysis of single cells at the gene level.

Topics: Adenocarcinoma; Adult; Aged; Bone Marrow Examination; Bone Marrow Neoplasms; Carcinoma, Squamous Cell; Esophageal Neoplasms; Fluorescent Antibody Technique, Indirect; Humans; Immunohistochemistry; Keratins; Microscopy, Confocal; Middle Aged; Plasminogen Activators; Predictive Value of Tests; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Sensitivity and Specificity; Stomach Neoplasms; Tumor Cells, Cultured

The detection of metastasizing single tumor cells has so far been difficult. Using a monoclonal antibody (MAb A45-B/B3) recognizing human cytokeratin, we identified immunocytochemically single tumor cells and micrometastases in patients (n = 24) with nonsmall cell lung cancer at the time of surgery of the primary tumor. The cytokeratin-positive cells (1-14/5 x 10(5) cells) in the bone marrow samples of 9 (9/24) patients were found. We also found a garland-like cluster, which consists of seven cancer cells and two closely connected tumor cells from one bone marrow sample. These results indicate that this technique can be used as a early diagnostic technique of bone marrow micrometastasis in the patient with the nonsmall cell lung cancer.

Topics: Animals; Antibodies, Monoclonal; Bone Marrow Neoplasms; Carcinoma, Non-Small-Cell Lung; Epitopes; Humans; Hybridomas; Immunohistochemistry; Keratins; Lung Neoplasms; Mice

It recently became evident that isolated tumor cells undetectable by conventional tumor staging are frequently present in bone marrow of patients with apparently localized non-small cell lung cancer (NSCLC). The clinical relevance of this minimal hematogenous tumor cell dissemination is under vigorous debate.. For tumor cell detection in the bone marrow, we used monoclonal antibody CK2 against the epithelial intermediate filament protein cytokeratin 18. The influence of a positive bone marrow finding on clinical outcome was studied in 139 patients with NSCLC postoperatively staged as pT1-4, pN0-2, M0, and R0 after a median follow-up of 66 months (range 48 to 74 months).. Cytokeratin-18-positive cells in bone marrow were demonstrated in 83 (59.7%) patients at the time of primary surgery and in 6 of 12 representative patients analyzed twice 3 to 18 months after surgery. In patients without histopathological lymph node metastases (pN0; n = 66), the occurrence of 2 or more tumor cells in bone marrow at primary surgery was a strong and independent predictor for overall survival (p = 0.007) in univariate analysis. The multivariate analysis showed a 2.8 times increased risk for shorter survival in patients with disseminated tumor cells versus patients without such cells. Four of the 6 patients with a positive cytokeratin status after surgery developed a tumor recurrence 11 to 44 months after the operation, while none of the patients with a negative bone marrow at all time intervals showed a tumor relapse.. Minimal residual bone marrow involvement is an independent prognostic factor for overall survival in patients with node-negative NSCLC, which may help to identify patients in need of an adjuvant systemic therapy. The postoperative persistence or reappearance of tumor cells in bone marrow indicates that these are not only shed cells but rather represent true micrometastasis.

Topics: Bone Marrow; Bone Marrow Neoplasms; Carcinoma, Non-Small-Cell Lung; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Lymph Nodes; Lymphatic Metastasis; Middle Aged; Multivariate Analysis; Risk Factors; Survival Rate

We looked for disseminated tumour cells postoperatively in the bone marrow of 22 patients with gastric cancer and of 26 patients with colorectal cancer. On an average the follow-up was 6.5 months in the gastric cancer group and 9 months in the colorectal cancer group. In 27% of the gastric cancer group and in 30% of the colorectal cancer group follow-up aspirates were obtained. Micrometastases were detected in 96% of colorectal cancer patients and in 77% of gastric cancer patients. Both, postoperative and follow-up aspirations showed clear differences in the number of ck+ cells between the iliac crest sides. The majority of cells formed clusters from 2 to about 200 cells. In both groups no significant correlation was found between the number of tumour cells detected and the established risk factors (stage, tumour extension, lymph node involvement, distant metastasis) or the disease-free survival. In the gastric cancer group higher numbers of ck+ cells were seen in the low tumor stages I and II, as compared to stage III and IV. Besides, in this group significantly more ck+ cells were detected in younger patients (age under 63,5) than in the older group over 63,5 years. Colorectal cancer patients did not show this correlation. In follow-up aspirations the number of ck+ cells of the colorectal and also the gastric cancer group showed a tendency of rise in relapse and of fall in disease-free survival. There was a tendency of higher rates of ck+ cells in the colorectal cancer group when compared to gastric cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Marrow Cells; Bone Marrow Examination; Bone Marrow Neoplasms; Colorectal Neoplasms; Disease Progression; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Keratins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prospective Studies; Risk Factors; Statistics as Topic; Stomach Neoplasms

In a subset of patients with early gastric cancer, there were recurrences of the disease after a curative resection had been done. Direct evidence of tumor seeding in distant organs at the time of surgery for gastric cancer is not available. An immunocytochemical assay for epithelial cytokeratin protein may fill this gap because it is a feature of epithelial cells that would not normally be present in bone marrow. From 1994-1997, the bone marrow of 45 patients with early gastric cancer was examined for tumor cells, using immunocytochemical techniques and an antibody reacting with cytokeratin, a component of the intracytoplasmic network of intermediate filaments. Intratumoral microvessels were stained with anti-CD31 monoclonal antibody. Clinicopathological characteristics were determined for subjects with cytokeratin-positive cells in the bone marrow. Of these 45 patients, 9 (20.0%) had cytokeratin-positive cells in the bone marrow at the time of primary surgery. These positive findings were not related to tumor advance-related factors of lymph node metastasis and distinct lymphatic and vascular invasion. Microvessel density in the primary tumor exceeded 2-fold in cytokeratin-positive cells, compared with findings in negative cells (P < 0.05). Tumor cells in bone marrow are indicative of the general disseminative metastasis in patients with early gastric cancer, and the metastatic potential was closely related to angiogenesis in the primary tumor.

Topics: Aged; Antibodies, Monoclonal; Bone Marrow Neoplasms; Female; Gastric Mucosa; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Stomach Neoplasms

The purpose of this study was to assess the immunocytochemical status of bone marrow aspirates from patients with clinically isolated hepatic metastases to test the hypothesis that such findings would allow improved patient selection for liver-directed treatment.. All patients had biopsy-proven or presumed colorectal cancer metastatic to the liver and were scheduled for an operative procedure for hepatic resection or for hepatic artery catheter and chemotherapy pump implant. Immunocytochemical analysis of bone marrow aspirate smears was performed with a panel of monoclonal antibodies directed toward cytokeratins, Lewis Y antigen and A-33 colorectal epitopes.. Data from 80 patients indicated that bone marrow reactivity was present in 9.5 percent of those with resectable hepatic metastases and in 34 percent of those not resected (P = 0.03). No single monoclonal antibody or combination produced better discrimination.. Presence or absence of presumed occult colorectal cancer cells in the bone marrow of patients with isolated hepatic metastases is biologically interesting, but not useful in selecting or altering patient management.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Bone Marrow; Bone Marrow Examination; Bone Marrow Neoplasms; Colorectal Neoplasms; Combined Modality Therapy; Humans; Immunoenzyme Techniques; Keratins; Lewis Blood Group Antigens; Liver; Liver Neoplasms; Prognosis

Detection of isolated tumour cells (TCs) in bone marrow (BM) from epithelial cancer patients by immunocytochemical (ICC) analysis seems to predict future relapse, but the reported percentages of positive BMs among patients with localized cancer show large variations and the number of detected TCs is low. This emphasizes the importance of thoroughly testing the methods in use. This study was performed to clarify to what extent positive staining of haematopoietic cells (HCs) interferes with the ICC detection of epithelial cells in BM. BM mononuclear cells (MNCs) from normal donors and stage I-II breast cancer patients were stained with anti-cytokeratin (CK) and isotype control monoclonal antibodies (MAbs) followed by alkaline phosphatase (AP)-based visualization of immunolabelled cells. In the ICC staining of normal donors by the anti-CK MAbs AE1/AE3 or A45-B/B3, rare immunoreactive cells were detected in 7/20 and 8/19 BMs, respectively. Morphological examination recognized all these cells as typical HCs. In the breast cancer patients (n = 257), anti-CK-positive cells were detected in 26.6 per cent, excluding cells with HC morphology. Using the same morphological criteria, isotype control-positive cells were detected in 5.4 per cent of patients. Some of the false-positive events were further analysed and cells with strong reactivity against the AP enzyme alone were detected. Double ICC staining recognized the majority of these AP directly-reactive cells as CD45-negative and human Ig kappa/lambda-positive, in accordance with the phenotype of mature plasma cells. Morphological evaluation and adequate controls are important to ensure the diagnostic specificity of micrometastases in BM. It is recommended that the number of BM MNCs included in negative controls should equal the number of cells in the diagnostic specimens.

Topics: Alkaline Phosphatase; Antibodies, Monoclonal; Bone Marrow Neoplasms; Breast Neoplasms; Epithelial Cells; False Positive Reactions; Female; Humans; Immunoenzyme Techniques; Keratins; Plasma Cells; Staining and Labeling

There is considerable interest in an autologous transplantation (AT) programme for patients with high-risk breast cancer; however, the issue of the incidence of occult bone marrow (BM) micrometastasis at diagnosis, and the cancer contamination of peripheral blood stem cell (PBSC) collections used for haematological rescue, is still debated. The presence of BM micrometastasis was evaluated in bilateral BM biopsies obtained at diagnosis of 33 patients with stage II/IIIA breast cancer using: (i) a 'nested' reverse transcriptase-polymerase chain reaction (RT-PCR) assay for cytokeratin 19 (K19) mRNA, (ii) histology, and (iii) immunohistochemistry (IHC) analysis with a panel of three monoclonal antibodies. The RT-PCR assay only was used to determine contamination of PBSC collections obtained after priming with recombinant human granulocyte-colony stimulating factor (rhG-CSF). K19 transcripts in one or both BM samples were detected in 48% of patients at diagnosis, with an overall 85% concordance with the results of IHC analysis. On the other hand, 56% of PCR- and IHC-positive BM samples were diagnosed as 'normal' on histological analysis. 57% of patients showed K19 mRNA in at least one PBSC collection; the possibility to have contaminated PBSC collections was significantly higher in patients with K19 positivity in BM at diagnosis. In four patients who had shown K19 positivity in BM and in PBSC collections, immunoselected CD34+ cells used for haematological rescue were K19-negative. There was a trend towards longer relapse free survival (RFS) in patients transplanted with K19-negative PBSC collections as compared to the others. In conclusion, a substantial proportion of patients with high-risk non-metastatic breast cancer present occult BM micrometastasis at diagnosis and also show cancer contamination of PBSC collections used for AT. These might represent a category of patients with poorer prognosis after AT, and possible candidates for more intensive and/or alternative therapeutic regimens, including AT with purged PBSCs.

Topics: Adult; Antigens, CD34; Bone Marrow Neoplasms; Breast Neoplasms; Disease-Free Survival; Female; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cell Transplantation; Humans; Immunohistochemistry; Keratins; Leukapheresis; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Treatment Outcome

Phenotyping of cytokeratin (CK)18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15-80% of cases and red CK18 staining in almost 100% of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (10(6) bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1-280 in 10(6); double staining 808, range 0-253) demonstrated relative reproducibility of APAAP results by double staining of 97%. Correlation of results between both methods was significant (p < 0.001, linear regression). Sensitivity of double staining tested in logarithmic tumor cell dilutions was one CK18-positive cell in 300,000. Specific uPA-R staining was seen on CK18-positive cells in bone marrow from 29 of 61 patients, and also on single surrounding bone marrow cells. To test the specificity of this staining, bone marrow cytospins from 10 patients without tumor disease were stained for uPA-R with the APAAP method. uPA-R expression was confirmed in all 10 cases, with a mean of 6.5% uPA-R-positive cells in 1000 bone marrow cells (SEM 1.2%). These results suggest that our double staining protocol is a sensitive, reproducible, and specific method for routine uPA-R phenotyping of disseminated CK18-positive cells in bone marrow of carcinoma patients.

Topics: Alkaline Phosphatase; Bone Marrow Neoplasms; Enzyme Precursors; Humans; Keratins; Phenotype; Plasminogen Activators; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Sensitivity and Specificity; Staining and Labeling; Stomach Neoplasms; Urokinase-Type Plasminogen Activator

Micrometastases within bone marrow have been shown to indicate a poor prognosis in patients with epithelial tumours. However, the degree to which micrometastases represent true residual disease or cell shedding and metastatic potential, is unclear.. To explore whether micrometastases represent residual disease, bone marrow taken from carefully staged patients before and after (> 6 months) "curative" resection of a primary gastrointestinal cancer was studied prospectively.. Seventy two consecutive patients were studied; the only exclusions were patients with known overt metastatic disease at the time of surgery. Micrometastatic cells were quantified per 10(5) marrow cells by flow cytometry after staining for contaminant cytokeratin-18 positive cells.. Micrometastases were detected preoperatively in 22% (16/72) of all patients, comprising 11 (23%) of 48 with colorectal cancer, five (33%) of 15 with gastric adenocarcinoma and none (0%) of nine with oesophageal squamous cancer. Although fewer metastatic cells were detected in postoperative bone marrow, and clearance of marrow deposits was evident in most patients, the persistence of micrometastases in five of 16 patients after resection, without evidence of tumour recurrence, indicates a subset with true residual disease. Detection of micrometastases postoperatively (persistent or newly developed) was significantly associated with development of overt metastases during the subsequent 12-18 months of follow up (nine of 19 patients) when compared with patients testing negative for micrometastases (eight of 53; p < 0.01).. Preoperative detection of micrometastases may reflect either transient shedding of cells, metastatic potential or residual disease, but postoperative micrometastases indicate minimal residual disease. Identification of these patients is important because they may benefit from adjuvant therapy.

Topics: Biomarkers, Tumor; Bone Marrow; Bone Marrow Neoplasms; Flow Cytometry; Gastrointestinal Neoplasms; Humans; Keratins; Neoplasm Staging; Neoplasm, Residual; Prognosis; Prospective Studies

This study was designed to evaluate the incidence and clinical implications of detection of micrometastatic cancer cells in bone marrow aspirates of patients with operable non-small cell lung cancer. The relationship between micrometastatic cells and p53 overexpression in the primary tumor was also assessed.. Thirty-nine patients with stages I through III non-small cell lung cancer who underwent curative resection were entered into this study. Immunohistochemistry with monoclonal antibody to cytokeratin 18 was used to detect tumor cells in bone marrow. Immunostaining of p53 protein in the corresponding primary tumors was also done.. Cytokeratin 18-positive cells were detected in 15 (39%) of the 39 patients. Overexpression of p53 was associated with positivity of the tumor cells in the bone marrow at borderline significance (14/29 versus 1/10; p = 0.0574). The patients with cytokeratin 18-positive cells in bone marrow demonstrated a significantly earlier recurrence than those without such cells (p = 0.0083, log-rank test).. Micrometastatic cancer cells were frequently present in bone marrow of patients with operable non-small cell lung cancer and may be a significant predictor of early recurrence. Further evaluation of this method may be useful in identifying patients with non-small cell lung cancer who are most likely to benefit from adjuvant chemotherapy.

Topics: Aged; Biomarkers, Tumor; Bone Marrow Neoplasms; Carcinoma, Non-Small-Cell Lung; Female; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Male; Middle Aged; Prognosis; Tumor Suppressor Protein p53

The aim of this study was to determine whether reverse transcriptase polymerase chain reaction (RT-PCR) for keratin 19 (K19) provides additional information when combined with immunohistochemistry when used to detect micrometastases in blood and bone marrow in patients with primary breast cancer. We studied 78 patients with breast cancer who had no evidence of distant metastases. We collected blood and bone marrow, separated the mononuclear fraction and carried out RT-PCR and immunohistochemistry for K19. RT-PCR was done by two 40-cycle rounds using nested primers. In initial experiments, RT-PCR was shown to be capable of detecting one tumour cell in one million normal bone marrow cells, which was at least 10 times more sensitive than immunohistochemistry, while retaining specificity. Five per cent of the peripheral blood and 22% of the bone marrow samples contained K19 positive cells by immunohistochemistry staining. Using RT-PCR, these proportions increased to 25% and 35%, respectively. This represents a significantly greater detection frequency (P < 0.001 and P = 0.03, respectively). RT-PCR for K19 is a more sensitive method for detecting micrometastases in patients with primary breast cancer when compared with immunohistochemistry.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Biomarkers, Tumor; Bone Marrow; Bone Marrow Neoplasms; Breast Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Middle Aged; Neoplasm Metastasis; Neoplastic Cells, Circulating; Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity

The present study evaluates the clinical significance of detection of cytokeratin 19 (K19) in the bone marrow of patients with breast cancer undergoing high-dose chemotherapy (HDCT) and autologous bone marrow transplantation (ABMT).. We studied retrospectively cryopreserved bone marrow aspirates from 83 patients with high-risk stage II, III, and IV breast cancer obtained before bone marrow harvest but after induction chemotherapy. All samples were histologically negative for metastases. Polymerase chain reaction (PCR) for K19 was performed according to methods described previously and results were correlated with the probability of relapse following HDCT and ABMT.. The incidence of occult metastases as defined by PCR for K19 message was 52% for 19 stage II, 57% for 14 stage III, and 82% for 50 stage IV patients (two-tailed P = .0075, chi 2 test). The probability of relapse at 3 years after ABMT was 32% and 94% for K19-positive stage II/III and stage IV patients, respectively, versus 10% and 14% for K19-negative stage II/III and stage IV patients, respectively. The difference was significant for stage IV patients (two-tailed P = .0002).. It has been shown that PCR is a highly sensitive method to detect K19 message in the bone marrow. The incidence of K19 positivity in bone marrow increases significantly with advancing stage. In patients with breast cancer, especially metastatic breast cancer, undergoing HDCT and ABMT, the presence of K19 is associated with a poor prognosis.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Neoplasms; Bone Marrow Transplantation; Breast Neoplasms; Carboplatin; Combined Modality Therapy; Disease-Free Survival; Etoposide; Female; Humans; Ifosfamide; Immunohistochemistry; Keratins; Middle Aged; Polymerase Chain Reaction; Prognosis; Recurrence; Retrospective Studies; Sensitivity and Specificity

In the present study, epithelial cells in the bone marrow of 42 patients with pancreatic carcinoma were identified immunocytochemically with monoclonal antibodies (MAbs) CK2, KL1 and A45-B/B3 directed to epithelial cytokeratins (CK), using the alkaline phosphatase anti-alkaline phosphatase method. The specificity of the MAbs was demonstrated by negative staining of marrow from 25 non-carcinoma age-matched control patients. Analysis of bone marrow aspirates from cancer patients revealed CK-positive cells in 14 (58.3%) of 24 cancer patients treated with curative intent and 10 (55.6%) of 18 patients with extended disease. After a median follow-up of 15.6 months (range 3-31 months), 5 (35.7%) out of 14 patients who underwent complete surgical resection but had tumour cells in bone marrow presented with distant metastasis and 6 (42.9%) with local relapse as compared to none of 10 corresponding patients without such cells (P < 0.05). The described technique may help to identify patients with pancreatic cancer and potential high risk of early metastatic relapse. The results promise to be of important assistance in determining prognosis and consequences in therapy of early stage pancreatic cancer.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Bone Marrow Neoplasms; Follow-Up Studies; Humans; Immunoenzyme Techniques; Keratins; Neoplasm Proteins; Neoplasm Recurrence, Local; Pancreatic Neoplasms; Prospective Studies

Minimal residual disease in patients with operable pancreas carcinoma is frequently missed by current non-invasive tumor staging.. We applied an immunocytochemical cytokeratin assay that allows the identification of individual pancreas carcinoma cells disseminated to bone marrow.. Prior to therapy bone marrow was aspirated from the upper iliac crest of 42 patients with pancreas carcinoma and a control group of 30 non-carcinoma patients. Tumor cells in cytologic bone marrow preparations were detected with monoclonal antibodies (mAbs) CK2, KL1 and A45-B/B3 to epithelial cytokeratins (CK), using the APAAP-method.. CK-positive cells were found in 14 (58.3%) of 24 cancer patients treated in curative intent and 10 (55.6%) of 18 patients with extended disease. After a mean follow up of 12.7 (3-32) months, 6 (42.8%) out of 14 patients who underwent complete surgical resection presented with tumor relapse and 5 (35.7%) with distant metastases as compared to none of 10 corresponding patients without such cells (p < 0.04). Moreover, patients with epithelial tumor cells in bone marrow showed also a significantly shorter overall survival than those without tumor cells (p < 0.03).. Immunocytochemical screening for epithelial tumor cells in bone marrow might contribute to an improved staging and is of prognostic relevance for pancreas carcinoma patients.

Topics: Adenocarcinoma; Biomarkers, Tumor; Biopsy, Needle; Bone Marrow; Bone Marrow Neoplasms; Follow-Up Studies; Humans; Immunoenzyme Techniques; Keratins; Pancreas; Pancreatectomy; Pancreatic Neoplasms; Survival Rate

Approximately half of the patients diagnosed with localized esophageal cancer die of metastatic disease within the first 2 years following tumor resection. The development of monoclonal antibodies (MAbs) directed against epithelial cell-associated and tumor antigens has enabled the detection of single disseminated tumor cells in secondary organs.. We used MAbs directed against epithelial cell antigens (i.e., cytokeratins) to determine the proportion of patients with esophageal cancer who display isolated tumor cells in their bone marrow. In addition, we evaluated the prognostic significance of a finding of bone marrow tumor cells in patients with esophageal cancer whose tumors were completely resected.. Prior to the initiation of treatment, bone marrow was aspirated from both sides of the upper iliac crests of 90 patients with squamous cell carcinoma of the esophagus. Bone marrow was also obtained from a population of 30 individuals who had not been diagnosed with cancer. Tumor cells in cytologic bone marrow preparations were detected by use of an assay that employed the MAbs CK2 (directed against cytokeratin 18), KL1 (directed against a 56,000-kd pan-cytokeratin component), and A45-B/B3 (directed against an epitope common to cytokeratins 8, 18, and 19) plus the alkaline phosphatase anti-alkaline phosphatasestaining method. Bone marrow biopsies, for conventional histologic examination with Giemsa staining, were performed on 62 patients. The Kaplan-Meier method and the logrank test were used to assess disease-free and overall survival according to the presence or absence of tumor cells in the bone marrow of 42 patients with completely resected tumors. Reported P values are two-sided.. Cytokeratin-positive tumor cells were detected in the bone marrow of 37 (41.1%) of the 90 total patients. The number of tumor cells detected per 10(5) mononuclear bone marrow cells ranged from one to 82. No significant differences in the numbers of disseminated tumor cells were noted for patients diagnosed with tumors at different stages. Only two (3.2%) of 62 bone marrow specimens examined after Giemsa staining showed morphologically identifiable tumor cells. Tumor cells were not detected in the bone marrow of patients who had not been diagnosed with cancer. After a median follow-up of 15.5 months (range, 6-33 months), 15 (79.0%) of 19 patients with completely resected tumors and tumor cells in their bone marrow had relapses compared with three (13.0%) of 23 patients with completely resected tumors and no tumor cells in their bone marrow (P = .019, logrank test). Patients with completely resected tumors and tumor cells in their bone marrow had significantly shorter overall survival than corresponding patients without tumor cells in their bone marrow (P = .036, logrank test).. Dissemination of esophageal cancer cells to the bone marrow is more frequent than expected from the rare occurrence of overt skeletal metastases. In general, the presence of tumor cells in the bone marrow may be an indicator of the disseminatory potential of individual tumors.

Topics: Antibodies, Monoclonal; Azure Stains; Bone Marrow Neoplasms; Carcinoma, Squamous Cell; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Survival Analysis

Up to 60% of patients with clinically localized prostate cancer will relapse despite potentially curative local treatment. Current staging tests have been limited in adequately identifying individual patients who are at a high risk for future relapse. Detection of bone marrow micrometastases may identify individuals destined to develop clinically detectable systemic metastases. Although immunohistochemistry and molecular approaches are being investigated, the most ideal test(s) are yet to be determined. In this report we describe methods for specific detection and isolation of prostate cancer micrometastases by multi-parameter rare event flow cytometric analysis. A model was developed and validated using three human prostate cancer cell lines, healthy donor marrow, dual marker labeling for cytokeratin (epithelial-specific marker) and CD45 (bone marrow-specific marker). The detection sensitivity of this model was at the level of one prostate cancer cell in 100,000 nucleated bone marrow cells. As a part of an ongoing clinical study, bone marrow aspirates from 15 patients with newly diagnosed prostate cancer have been analyzed. Six patients were found to have cytokeratin positive/CD45 negative cells in their bone marrow aspirates. We conclude that flow cytometric rare event analysis provides a sensitive and specific assay for detection of bone marrow micrometastases in patients with clinically localized prostate cancer.

Topics: Bone Marrow Neoplasms; Flow Cytometry; Humans; Keratins; Leukocyte Common Antigens; Male; Neoplasm Recurrence, Local; Pilot Projects; Prostatic Neoplasms; Specimen Handling; Tumor Cells, Cultured

A two-step reverse-transcriptase-based polymerase chain reaction (PCR) with nested primer pairs was developed to amplify sensitive and specific cytokeratin-19 (CK-19) mRNA sequences from human breast cancer cells. No CK-19 pseudogene interference was seen. The larger DNA-derived amplification products could be clearly discriminated from mRNA-derived products. The CK-19 message was not amplified from bone marrow or blood of healthy volunteers and patients with haematological malignancies nor from myeloid and lymphoid cell lines. Breast cancer cells were diluted in buffy coat cells up to 10(-6) and CK-19 mRNA sought by PCR. The CK-19 message was detected in 14 of 26 blood samples and 14 of 24 marrow samples but in neither of two peripheral blood stem cell samples taken from 35 breast cancer patients. By sequence-analysis control of two of these samples and two cell lines, the amplified DNA fragments were confirmed to be homologous with the CK-19 sequence. The CK-19 message was further sought in matched blood/marrow samples taken from 13 untreated women in the same cohort at the time of diagnosis. In 3 of these, CK-19 RNA was detected in blood and marrow and, in 3 others, only in blood, but never in marrow alone. The results show that CK-19 assay by reverse transcriptase/PCR is a sensitive and specific technique for the detection of cancer cells in bone marrow and blood. It could be helpful in diagnosis and monitoring of metastatic breast cancer and detection of micrometastases. This should be evaluated on larger numbers of patients, with different clinical samples and epithelial malignancies.

Topics: Base Sequence; Bone Marrow; Bone Marrow Neoplasms; Breast Neoplasms; Consensus Sequence; Female; Gene Expression; Humans; Keratins; Molecular Sequence Data; Neoplasm Metastasis; RNA, Messenger; RNA, Neoplasm; Sequence Alignment; Tumor Cells, Cultured

The usefulness of routine pancytokeratin staining of bone marrow cores (BMCs)--negative for hematoxylin and eosin (HE)--from patients with lobular carcinoma of the breast has been fairly well established. However, the role of wide-spectrum cytokeratin staining of HE-negative BMCs from patients with ductal carcinoma has not been as well determined and is the purpose of this study. Forty-two prospectively and retrospectively reviewed He-negative BMCs from 21 patients with ductal breast carcinoma failed to reveal staining with a wide-spectrum cytokeratin stain, supporting the selective use of this immunostain in "suspicious" cases with atypical cells detected in HE BMCs.

Topics: Biomarkers, Tumor; Bone Marrow; Bone Marrow Neoplasms; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Humans; Immunoenzyme Techniques; Keratins; Prospective Studies; Retrospective Studies; Staining and Labeling

It is unclear whether disseminated tumour cells detected in bone marrow in early stages of solid cancers indicate a subclinical systemic disease component determining the patient's fate or simply represent mainly irrelevant shed cells. Moreover, characteristics differentiating high and low metastatic potential of disseminated tumour cells are not defined. We performed repeated serial bone marrow biopsies during follow-up in operated gastric cancer patients. Most patients with later tumour relapse revealed either an increase or a constantly high number of tumour cells. In contrast, in patients without recurrence, either clearance of tumour cells or negative or low cell counts were seen. Urokinase plasminogen activator (uPA)-receptor expression on disseminated tumour cells was significantly correlated with increasing tumour cell counts and clinical prognosis. These results demonstrate a systemic component in early solid cancer, indicated by early systemically disseminated tumour cells, which may predict individual disease development.

Topics: Bone Marrow; Bone Marrow Neoplasms; Follow-Up Studies; Gastrointestinal Neoplasms; Humans; Keratins; Neoplastic Cells, Circulating; Prognosis; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Tumor Cells, Cultured

Immunohistochemical detection of bone marrow micrometastases has been reported as a prognostic marker in colorectal cancer. The aims of this study were to evaluate the potential advantage of flow cytometry as an objective method of identifying and quantifying micrometastatic deposits within bone marrow and to determine the prevalence and quantity of micrometastases in patients undergoing surgery for gastrointestinal cancers.. Flow cytometry was first validated by a controlled "spike" experiment in which varying numbers of neoplastic epithelial cells were added to bone marrow, and cytometry was performed in a blinded fashion. Three neoplastic cell lines (colonic and esophageal) with varying degrees of expression of cytokeratin-18 were used. Epithelial cells were detected by dual staining with fluorescence-labeled, monoclonal anti-cytokeratin, and propidium iodide.. Cytometry reproducibly detected the presence of > or = 10 neoplastic cells per 10(5) marrow cells. Micrometastases were found in 20%-30% of patients undergoing potentially curative resection of colorectal and gastroesophageal adenocarcinomas. There was a trend toward increasing positivity for marrow deposits with advanced Dukes' staging of colorectal cancer.. Flow cytometric assessment of bone marrow is a reliable, objective, and quantitative method of detecting micrometastatic deposits found in a substantial subset of patients undergoing surgery for gastrointestinal adenocarcinomas.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Bone Marrow Neoplasms; Carcinoma, Squamous Cell; Colorectal Neoplasms; Esophageal Neoplasms; Flow Cytometry; Gastrointestinal Neoplasms; Humans; Keratins; Prognosis; Prospective Studies; Stomach Neoplasms

A large proportion of patients with operable lung carcinoma (no evidence of systemic spread of tumor) develop metastatic disease after primary therapy. More sensitive and specific methods are needed to identify patients at highest risk for recurrence who may benefit most from adjuvant therapy, while sparing those patients who do not require such treatment.. Using epithelial-specific monoclonal antibodies, the authors have developed an immunocytochemical assay capable of detecting as few as 2 lung cancer cells in 1 million bone marrow cells.. The assay was used to test the bone marrow (from resected ribs) of 43 patients with primary non-small cell lung carcinoma who showed no clinical or pathologic evidence of systemic disease.. Occult bone marrow micrometastases (BMMs) were detected in 40% of patients (17/43) with non-small cell lung cancer, including 29% (5/17) of patients with stage I or II disease and 46% of whom (12/26) had stage III disease. The median follow-up was 13.6 months. Patients with occult BMMs had significantly shorter times to disease recurrence compared with patients without BMMs (7.3 vs. > 35.1 months, p = 0.0009). Furthermore, for patients with stage I or II disease, the presence of occult BMMs was significantly associated with a higher rate of recurrence (p = 0.0004).. The detection of occult BMMs identifies patients with operable non-small cell lung carcinoma who are at significantly increased risk for recurrence, independent of tumor stage, and may be useful in evaluating patients for adjuvant treatment protocols.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Bone Marrow Neoplasms; Carcinoma, Non-Small-Cell Lung; Epithelium; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Neoplasm Recurrence, Local; Risk Factors; Survival Rate