Cancer Cells In Peripheral Blood Research Articles

Optomicrofluidic detection of cancer cells in peripheral blood via metabolic glycoengineering

We report optomicrofluidic detection of circulating tumor cells (CTCs) in a mixture of CTCs and peripheral blood mononuclear cells (PBMCs) by exploiting the difference in their cell metabolism.

Identification and Comprehensive Co-Detection of Necrotic and Viable Aneuploid Cancer Cells in Peripheral Blood.

Simple SummaryCirculating tumor cells (CTCs) and CD31+ circulating tumor endothelial cells (CTECs) constitute a unique pair of cellular circulating tumor biomarkers and play a crucial role in cancer metastasis and disease progression. Precise detection of live and necrotic aneuploid CTCs and CTECs in therapeutic cancer patients, though clinically highly demanded, has yet to be achieved in the field. The aim of this study is to develop a comprehensive strategy to effectively distinguish and co-detect viable and dead non-hematological cancer cells harboring aneuploid chromosomes and expressing various tumor markers. The innovative strategy developed in the present study will assist in an efficient assessment of therapy effectiveness, rapid detection of emerging treatment resistance and bringing in new insights into the comprehension of cancer cells in circulation.Aneuploid circulating tumor cells (CTCs, CD31−) and circulating tumor endothelial cells (CTECs, CD31+) exhibit an active interplay in peripheral blood, and play an essential role in tumorigenesis, neoangiogenesis, disease progression, therapy-resistant minimal residual disease (MRD), cancer metastasis and relapse. Currently, most CTC detection techniques are restricted to the indistinguishable quantification of circulating rare cells, including both necrotic and viable cells in cancer patients. Clinically imperative demands to distinguish and detect live and/or dead non-hematological aneuploid cancer cells in peripheral blood, which will assist in the rapid evaluation of therapeutic effects, real-time monitoring of treatment resistance longitudinally developed along with therapy and the effective detection of post-therapeutic MRD, have not yet been achieved. The integrated subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH)-derived novel strategy was developed in this study, aiming to precisely identify and detect live and necrotic cancer cells (NC) enriched from carcinoma patients’ biofluids. The innovative SE-iFISH (NC) provides a meaningful and practical approach to co-detect various viable and necrotic aneuploid CTCs and CTECs. The detected circulating rare cells can be characterized and categorized into diverse subtypes based upon cell viability, morphology, multiple tumor markers’ expression, and the degree of aneuploidy relevant to both malignancy and therapeutic resistance. Each subtype of live or necrotic CTCs and CTECs possesses distinct utility in anti-cancer drug development, translational research, and clinical practice.

An integrated enrichment system to facilitate isolation and molecular characterization of single cancer cells from whole blood.

Circulating tumor cells (CTCs) carry valuable biological information. While enumeration of CTCs in peripheral blood is an FDA-approved prognostic indicator of survival in metastatic prostate and other cancers, analysis of CTC phenotypic and genomic markers is needed to identify cancer origin and elucidate pathways that can guide therapeutic selection for personalized medicine. Given the emergence of single-cell mRNA sequencing technologies, a method is needed to isolate CTCs with high sensitivity and specificity as well as compatibility with downstream genomic analysis. Flow cytometry is a powerful tool to analyze and sort single cells, but pre-enrichment is required prior to flow sorting for efficient isolation of CTCs due to the extreme low frequency of CTCs in blood (one in billions of blood cells). While current enrichment technologies often require many steps and result in poor recovery, we demonstrate a magnetic separator and acoustic microfluidic focusing chip integrated system that enriches rare cells in-line with FACS™ (fluorescent activated cell sorting) and single-cell sequencing. This system analyzes, isolates, and index sorts single cells directly into 96-well plates containing reagents for Molecular Indexing (MI) and transcriptional profiling of single cells. With an optimized workflow using the integrated enrichment-FACS system, we performed a proof-of-concept experiment with spiked prostate cancer cells in peripheral blood and achieved: (i) a rapid one-step process to isolate rare cancer cells from lysed whole blood; (ii) an average of 92% post-enrichment cancer cell recovery (R2 = 0.9998) as compared with 55% recovery for a traditional benchtop workflow; and (iii) detection of differentially expressed genes at a single cell level that are consistent with reported cell-type dependent expression signatures for prostate cancer cells. These model system results lay the groundwork for applying our approach to human blood samples from prostate and other cancer patients, and support the enrichment-FACS system as a flexible solution for isolation and characterization of CTCs for cancer diagnosis. © 2018 International Society for Advancement of Cytometry.

A 2-Gene Panel Derived From Prostate Cancer-Enhanced Transcripts in Whole Blood Is Prognostic for Survival and Predicts Treatment Benefit in Metastatic Castration-Resistant Prostate Cancer.

To determine a prognostic model derived from prostate cancer-enhanced transcripts in whole blood of castration-resistant prostate cancer (CRPC) patients and explore its applicability as a surrogate of treatment response. Six out of twenty-three selected transcripts were identified as specific for detection of metastatic prostate cancer cells in peripheral blood using quantitative polymerase chain reaction (qPCR). Their prognostic value was explored in whole blood samples of a training cohort (n = 22 CRPC patients, New York, USA). A resulting 2-gene panel (2GP) including KLK2 and TMPRSS2 was validated in an independent cohort with pre- and post-treatment blood draws after 9-16 weeks of systemic treament (n = 86 CRPC patients, Munich, Germany). Overall survival (OS), prostate-specific antigen progression-free survival (PSA-PFS), and clinical PFS were analyzed. Kaplan-Meier and cox regression analyses were performed. An unfavorable 2GP (≥1 marker positive) identified patients with poor survival (median OS 10.0 months [95%CI 5.7-14.2] vs. not reached; P = 0.023). This was validated in an independent cohort at pre-treatment (median OS 7.8 [95%CI 6.5-9.2] vs. 17.3 months [95%CI 10.7-23.8]; P = 0.004) and post-treatment blood draw (median OS 5.0 [95%CI 0.0-10.0] vs. 18.0 months [95%CI 9.5-26.6]; P = 0.003). The 2GP independently predicted OS on multivariate analysis (hazard ratio 2.1 [95%CI 1.1-4.0]; P = 0.034) and performed better than PSA decline at correlation with OS. Conversion to favorable 2GP during treatment correlated with improved OS (7.8 to 20.9 months), PSA-PFS (2.8 to 12.0 months), and clinical PFS (4.6 to 8.0 months). The established 2GP is prognostic for survival at pre- and post-treatment blood draw in CRPC patients and conversion to favorable 2GP predicts treatment benefit. Prostate 76:1160-1168, 2016. © 2016 Wiley Periodicals, Inc.

English

Application of hemofiltercytological analysis shows its ample opportunities in assessment of polychemotherapy completeness and quality through the study of drug pathomorphosis. The objective of the study was development and studying of the clinical relevance of hemofiltercytological venous blood test as completeness and quality control of patients with colorectal cancer adjuvant chemotherapy. 35 of 39 tested cancer patients who underwent radical surgery for colorectal cancer of different localization, including cases of locally advanced stage had cancer cells in peripheral blood. Thus, the percentage of patients who had atypical cells in venous blood in postoperative period was 89.7. Adjuvant chemotherapy was offered to all 35 patients with diagnosed carcinemia and after their consent it was held in modes FOLFOX-4 and XELOX. Evaluation of anticancer drugs therapeutic action is traditionally carried out using the criteria of objective and subjective effects. The criterion of objective effect during chemotherapy of solid tumors is reduction of tumor and metastases and the criterion of subjective one is well-being of a patient. In addition, it is important to notice the highest drug resistance observed at rectum cancer, because there is a predominance of smaller stages I to II of drug pathomorphosis (9 to 7) over greater stages III-IV (7 to 1) among these cancer patients. Consequently, hemofiltercytological blood test provides a real opportunity to assess the effect of polychemotherapy and to solve the problem of chemotherapeutic treatment courses number necessary to stabilize the cancer process. Key words: Calibrated filter, hemofiltercytological blood test, carcinemia, microscreening.

High-purity separation of cancer cells by optically induced dielectrophoresis.

Detecting and concentrating cancer cells in peripheral blood is of great importance for cancer diagnosis and prognosis. Optically induced dielectrophoresis (ODEP) can achieve high resolution and low optical intensities, and the electrode pattern can be dynamically changed by varied light patterns. By changing the projected light pattern, it is demonstrated to separate high-purity gastric cancer cell lines. Traditionally, the purity of cancer cell isolation by negative selection is 0.9% to 10%; by positive selection it is 50% to 62%. An ODEP technology is proposed to enhance the purity of cancer cell isolation to about 77%.

Investigation of free cancer cells in peripheral blood using CEA mRNA expression in perioperative colorectal cancer patients

The aim of this study was to evaluate the impact of laparoscopic surgery (Lap) on circulating free tumor cells in colorectal cancer patients. In this study, we selected carcinoembryonic antigen (CEA) mRNA expression in peripheral blood as the marker of the circulating tumor cells and compared this marker between Lap and open colectomy (OC), to investigate differences due to surgical approach. A total of 50 patients underwent curative surgery for solitary colorectal cancer at our department, between June, 2008 and February, 2011. The patients were divided into OC and Lap groups (25 patients each). Total RNA was extracted subsequent to peripheral blood collection prior to surgery, immediately following surgery and 1, 3 and 7 days after surgery. CEA mRNA was detected with reverse transcription polymerase chain reaction (RT-PCR) and the association between peripheral blood CEA mRNA-positive rate, surgical findings and clinicopathological characteristics was investigated. The peripheral blood CEA mRNA-positive rate was significantly increased immediately after surgery, compared to the preoperative rate (P=0.001), but decreased over time. No significant differences were observed at any blood-sampling time point after postoperative day 1. The positive rate was significantly increased in the OC group immediately after surgery, compared to the preoperative rate (P=0.004). However, there were no significant differences between the rates prior to and immediately after surgery in the Lap group. The patients were then divided into those who were peripheral blood CEA mRNA-positive and -negative after surgery (postoperative positive and negative groups, respectively) and the clinicopathological characteristics were compared. Significant differences were identified between the groups in lower rectal cancer patients and patients with a large intraoperative blood loss (P=0.001 and P=0.01, respectively). In conclusion, in colorectal cancer patients, there were no significant differences in the perioperative peripheral blood CEA mRNA-positive rate or its short-term changes between patients undergoing OC and Lap surgery. It was suggested that Lap is equivalent to OC with regard to free cancer cells.

Detection of circulating tumor cells in peripheral blood from patients with gastric cancer using piRNAs as markers

ObjectiveTo investigate the feasibility of detecting circulating cancer cells in peripheral blood from gastric cancer patients using Piwi-interacting RNAs (piRNAs) as markers. Design and methodsReal-time quantitative reverse transcription-polymerase chain reaction was used to analyze piR-651 and piR-823 levels in the peripheral blood of 93 patients with gastric cancer and 32 healthy volunteers. Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic values. ResultsThe peripheral blood levels of piR-651 and piR-823 in the patients with gastric cancer were significantly lower than those from controls (P<0.001). The piR-651 level in gastric adenocarcinoma was higher than that in gastric signet ring cell carcinoma (P=0.003). The piR-823 level was positively associated with tumor-node-metastasis stage (P=0.027) and distant metastasis (P=0.026). The areas under the ROC curve were 0.841, 0.812 and 0.860 for piR-651, piR-823 and the combination, respectively. ConclusionspiRNAs may be valuable biomarkers for detecting circulating gastric cancer cells.

Detection of circulating tumor cells in peripheral blood from patients with gastric cancer using microRNA as a marker

Recently, the detection of occult cancer cells in peripheral blood has received a great deal of attention regarding the prediction of postoperative cancer recurrence and for novel strategies of adjuvant therapy. The aim of this study was to establish a new molecular diagnostic method of detecting circulating tumor cells. Gastric cancer SGC-7901 cells in 2 ml blood from healthy volunteers were serially diluted. Additional peripheral blood samples were collected from 90 patients and 27 healthy volunteers. Real-time reverse transcription-polymerase chain reaction was used to detect the levels of microRNA-106a (miR-106a) and microRNA-17 (miR-17). Receiver operating characteristics (ROC) curves were constructed. In recovery experiments, a significant correlation between the number of cancer cells and the levels of both miR-106a (r = -0.906, p = 0.037) and miR-17 (r = -0.912, p = 0.031) was found. In preoperative and postoperative patient groups, miR-106a and miR-17 levels were significantly higher than those in controls. The areas under the ROC curve for miR-106a, miR-17, and combination were 0.684 (p = 0.0066), 0.743 (p = 0.0001), and 0.741 (p = 0.0002), respectively. Our results indicate that the detection of miRNA in peripheral blood may be a novel tool for monitoring circulating tumor cells in patients with gastric cancers.

Peripheral thyrotropin receptor mRNA as a novel marker for differentiated thyroid cancer diagnosis and surveillance

Thyroid cancer is the most prevalent endocrine cancer whose incidence rates, particularly among women, have increased over the last decade. Although survival outcomes following surgery (with or without radioactive iodine ablation treatment) remain favorable, a significant proportion of patients are at lifetime risk of locoregional lymph node recurrence and distant metastasis. Serum thyroglobulin (Tg) has been the only circulating marker in routine use for detecting thyroid cancer recurrence, but it lacks sensitivity and is unreliable when Tg antibodies are present. New molecular markers for thyroid cancer have been investigated, with most based on detection in thyroid nodule or tumor tissue specimens. Recently, it has become possible to detect thyroid cancer cells in peripheral blood by measuring the mRNA of thyroid-specific genes, such as the mRNA of Tg and thyrotropin receptor. These have become promising new circulating markers for thyroid cancer. This review highlights the progress in this field from the perspective of improved initial cancer diagnosis and enhanced ability to monitor thyroid cancer recurrence.

Detection of mammaglobin mRNA in peripheral blood is associated with high grade breast cancer: Interim results of a prospective cohort study

BackgroundWe sought to examine the detection rate of cancer cells in peripheral blood (PBL) and in bone marrow (BM) using an established 7-gene marker panel and evaluated whether there were any definable associations of any individual gene with traditional predictors of prognosis.MethodsPatients with T1-T3 primary breast cancer were enrolled into a prospective, multi-institutional cohort study. In this interim analysis 215 PBL and 177 BM samples were analyzed by multimarker, real-time RT-PCR analysis designed to detect circulating and disseminated breast cancer cells.ResultsAt a threshold of three standard deviations from the mean expression level of normal controls, 63% (136/215) of PBL and 11% (19/177) of BM samples were positive for at least one cancer-associated marker. Marker positivity in PBL demonstrated a statistically significant association with grade II-III (vs. grade I; p = 0.0083). Overexpression of the mammaglobin (mam) gene alone had a statistically significant association with high tumor grade (p = 0.0315), and showed a trend towards ER-negative tumors and a high risk category. There was no association between marker positivity in PBL and the pathologic (H&E) and/or molecular (RT-PCR) status of the axillary lymph nodes (ALN).ConclusionThis study suggests that molecular detection of circulating cancer cells in PBL detected by RT-PCR is associated with high tumor grade and specifically that overexpression of the mam gene in PBL may be a poor prognostic indicator. There was no statistically significant association between overexpression of cancer-associated genes in PBL and ALN status, supporting the concept of two potentially separate metastatic pathways.

Real-time quantitative RT-PCR assay of prostate-specific antigen and prostate-specific membrane antigen in peripheral blood for detection of prostate cancer micrometastasis

Objective Reverse transcription-polymerase chain reaction (RT-PCR) was widely used to detect disseminated tumor cells. However, high percentages of false-positive cases have been reported. Herein, we developed a method to detect prostate cancer (PCa) micrometastasis by real-time quantitative RT-PCR. Methods Blood samples were obtained from 118 males, including 13 healthy volunteers, 17 patients with metastatic PCa, 20 patients with localized PCa, and 68 patients with BPH. RNA was isolated from blood samples of different patients. Real-time RT-PCR was used to quantify the copy number of PSA and PSMA. The primers and probe of PSMA were designed to specifically discriminate between PSMA and PSM′, an alternatively spliced variant, mRNA. Results There was significant difference in the PSA and PSMA mRNA levels among BPH, locally confined PCa, and metastatic PCa blood specimens. The real-time quantitative RT-PCR is sensitive, accurate, and has a high reproducibility within a wide dynamic range (10 3–10 8), which permits simultaneous quantitative analysis of samples with varying input concentrations. Conclusions This method improves accuracy and liability of assessment of disseminated prostate cancer cells in peripheral blood and is promising for clinical diagnosis purpose.

CK19 mRNA expression measured by reverse-transcription polymerase chain reaction (RT-PCR) in the peripheral blood of patients with non-small cell lung cancer treated by chemo-radiation: An independent prognostic factor

To investigate the prognostic significance of circulating cancer cells in peripheral blood (PB) of patients with non-small cell lung cancer (NSCLC) treated by chemo-radiation with curative intent. Cytokeratin-19 (CK19) mRNA was measured by nested reverse-transcription polymerase chain reaction (RT-PCR) in PB from 67 NSCLC patients before and after chemo-radiation. The measurements of CK19 mRNA were compared to the outcome of therapy to evaluate its significance of prognoses. The sensitivity and specificity of CK19 RT-PCR was 10(-7) and 96.7%, respectively. The positive rate of CK19 mRNA in PB before chemo-radiation was correlated only to N stage (p=0.014). In contrast, it was more closely correlated to histological types (adenocarcinoma versus non-adenocarcinoma) (p=0.019), weight loss (p=0.010), KPS status (p=0.027) and N stage (p=0.032) after chemo-radiation. CK19 status in PB before chemo-radiation did not permit predictions of overall survival (p=0.375) and progression-free survival (p=0.573). However, the patients with CK19 mRNA expression in PB after treatments had poorer overall survival (p<0.001) and progression-free survival (p<0.001) as compared to those with negative CK19 mRNA expression. The worst survivals were seen in patients with persistently positive CK19 mRNA expression both before and after treatments. Multivariate analyses demonstrated that the positivity of CK19 mRNA after chemo-radiation was an independent unfavorable prognostic factor for both overall survival and progression-free survival (p<0.001 and <0.001). Only after chemo-radiation could the measurement of CK19 mRNA in PB predict the prognoses of NSCLC. Patients with the positive CK19 mRNA had shorter survival compared to the negative patients.

AFP mRNA Detected in Bone Marrow by Real-Time Quantitative RT-PCR Analysis Predicts Survival and Recurrence After Curative Hepatectomy for Hepatocellular Carcinoma

To determine whether detection of hepatocellular carcinoma (HCC) cells by real-time quantitative RT-PCR targeting of alpha-fetoprotein mRNA (AFP mRNA) before or after curative hepatectomy predicts HCC recurrence and patient survival. The presence of cancer cells in peripheral blood and/or bone marrow in patients with malignant disease has been reported to correlate with outcome. Between July 2000 and June 2005, 136 consecutive HCC patients underwent primary curative hepatectomy. Bone marrow aspirated preoperatively, and peripheral blood samples collected before and after operation were subjected to real-time quantitative RT-PCR analysis using AFP mRNA as a target molecule. Median follow-up was 23 months (range, 6-54 months). Patient survival (PS), disease-free survival (DFS), and clinicopathologic features were compared between patients with positive and negative AFP mRNA. Twenty-four patients died (22 from HCC). HCC recurred in 66 patients (hepatic in 37 [56.1%]; hepatic and remote in 17 [25.8%], and remote alone in 12 [18.2%]). Bone marrow was positive for AFP mRNA in 38 patients (27.9%) and negative in 98 (72.1%). One- and 3-year PS was 96.6% and 91.4%, respectively, with negative AFP mRNA versus 86.2% and 55.5%, respectively, with positive AFP mRNA (P < 0.0001). One- and 3-year DFS were 73.2% and 44.8%, respectively, with negative AFP mRNA versus 54.5% and 25.8%, respectively, with positive AFP mRNA (P = 0.0399). Portal vascular invasion, tumor size, multiple tumors, and tumor differentiation correlated with inferior PS and DFS on univariate analysis. On multivariate analysis, positive AFP mRNA was the most important risk factor for PS (P = 0.001) and DFS (P = 0.0165). In addition, positive AFP mRNA in peripheral blood after operation tended to predict reduced DFS. AFP mRNA in the bone marrow and systemic circulation during the perioperative period predicts patient survival and recurrence after curative hepatic resection for HCC.

Microsystems for isolation and electrophysiological analysis of breast cancer cells from blood

This paper presents the development of a microsystem for separating suspended breast cancer cells in peripheral blood and for sorting them based on their electrophysiological characteristics. A continuous paramagnetic capture mode (PMC) magnetophoretic microseparator was utilized for the isolation of suspended breast cancer cells in peripheral blood based on the native magnetic properties of blood cells without any tagging such as with magnetic probes. A micro-electrical impedance spectroscopy (mu-EIS) system was used as a downstream cell analysis tool to extract the pathological characteristics from the breast cancer cells. The system was fabricated on silicon and glass substrates utilizing microfabrication and stereolithography technologies. The experimental results of the PMC microseparator show that 94.8% of the breast cancer cells could be continuously separated out from a spiked blood sample with a 0.2 T external magnetic flux. The electrical impedances of human breast cancer cell lines of different pathological stages (MCF-7, MDA-MB-231, and MDA-MB-435) were measured using mu-EIS and compared to those of normal human breast tissue cell line MCF-10A.

Specific Detection of Cytokeratin 20-Positive Cells in Blood of Colorectal and Breast Cancer Patients by a High Sensitivity Real-Time Reverse Transcriptase-Polymerase Chain Reaction Method

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (single-round RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P = 0.026) but not colorectal cancer patients (P = 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method.

Detection of circulating cancer cells in peripheral blood as a prognostic factor in early breast cancer

Detection of circulating cancer cells in peripheral blood as a prognostic factor in early breast cancer

Detection of minimal disease in breast cancer

We investigated the prognostic significance of circulating breast cancer cells in peripheral blood detected by quantitative RT-PCR of marker genes in patients with advanced breast cancer. Blood samples from 94 breast cancer patients with metastatic disease (M1) were examined for circulating tumour cells by studying the mRNA expression of CK19, p1B, PS2, EGP2, mammaglobin and SBEM by real-time PCR. Using a score function, developed for predicting circulating tumour cells by quadratic discriminant analysis (QDA), four expression levels were combined into a single discriminant value. Tumour cells were present in 24 out of 94 (31%) of the patients, as compared with 0 out of 104 controls. The patients with a positive QDA value did have a progression-free survival at 1 year of 3% and overall survival at 2 years of 17%, against 22% and 36% for patients with a negative QDA value (P = 0.015 and P = 0.0053, respectively). Breast cancer patients with metastatic disease have a significantly worse progression-free survival and overall survival when circulating tumour cells can be detected in their peripheral blood [1]. This method was used but was not sensitive enough to predict survival on the basis of a positive score in peripheral stem cell preparations (PBSC) of patients treated in a high-dose chemotherapy trial. The number of tumour cells in these preparations is too low. We therefore sought a method that would specifically select breast tumour cells. We now show that a combination of magnetic beads cell separations and one-step cDNA synthesis of mRNA increases the sensitivity by at least 10-fold. Ten MCF7 cells mixed into 10 ml peripheral blood of a healthy control can be easily identified. To validate this new method we will analyze blood samples of 200 stage I or stage II breast cancer patients and 50 PBSC of breast cancer patients (and 20 PBSC of patients with lymphoid malignancy as controls). So far, 30 stage I or stage II patients have been evaluated with the marker panel of six genes. A combination of four markers employed in the discriminant score revealed a sensitivity of 10% in detecting epithelial marker genes in peripheral blood. The cell separation technique allows at least a 10-fold increase in sensitivity to detect tumour cell specific mRNA in mixed cell suspensions. Further analysis of the 50 PBSC patients and the prospective 200-patient stage I and stage II diagnostic study will reveal specificity and sensitivity. Interim results indicate an increase in sensitivity of at least 10%.

Mammaglobin Remains a Useful Marker for the Detection of Breast Cancer Cells in Peripheral Blood

Mammaglobin Remains a Useful Marker for the Detection of Breast Cancer Cells in Peripheral Blood

Detection of occult cancer cells in peripheral blood and bone marrow by quantitative RT-PCR assay for cytokeratin-7 in breast cancer patients

The clinical significance of occult micrometastasis (O.M) remains unknown. We investigated it in peripheral blood (P.B.) and bone marrow (B.M.) in breast cancer patients with surgery. First, we investigated the expression levels of 7 representative molecular markers for detecting O.M (CEA, CK-7, CK-18, CK-19, CK-20, MAM and MUC-1) in 27 cancer and 8 non-epithelial cell lines using quantitative RT-PCR (QRT-PCR), and showed that the expression level of CK-7 was higher in every cancer cell line than in the non-epithelial cell lines. Next, we studied the clinical significance of O.M in P.B. and B.M. by QRT-PCR for CK-7 in breast cancer patients with surgery. Based on comparison with 17 non-cancer controls, 37 (18.0%) and 100 (48.5%) of the 206 patients were positive for CK-7 in P.B. and B.M., respectively. In 98 cases observed over 24 months after surgery, the CK-7-positive group in P.B. had poorer disease-free survival (DFS) than the negative group (p<0.01). The CK-7-positive group in P.B. showed poorer DFS than the negative group in 132 lymph node-negative cases (p=0.01), and moreover, in 61 lymph node-negative cases observed over 24 months after surgery, the CK-7-positive group in P.B. showed poorer DFS than the negative group (p<0.0001). In B.M., no significant difference in DFS was found between the CK-7-positive and CK-7-negative groups. QRT-PCR for CK-7 could be a useful and universal method for detecting O.M, and the quantitative detection of CK-7 in P.B. would have a prognostic value as a marker of early recurrence in breast cancer patients with surgery.