S. Kaul

Multimarker Gene Analysis of Circulating Tumor Cells in Pancreatic Cancer Patients: A Feasibility Study

Objective: The aim of this study was to develop an immunomagnetic/real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and assess its clinical value for the molecular detection of circulating tumor cells (CTCs) in peripheral blood of pancreatic cancer patients. Methods: The presence of CTCs was evaluated in 34 pancreatic cancer patients before systemic therapy and in 40 healthy controls, through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 [targeting mucin 1 and epithelial cell adhesion molecule (EpCAM), respectively], followed by real-time RT-PCR analysis of the genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. Results: The developed assay showed high specificity, as none of the healthy controls were found to be positive for the multimarker gene panel. CTCs were detected in 47.1% of the pancreatic cancer patients before the beginning of systemic treatment. Shorter median progression-free survival (PFS) was observed for patients who had at least one detectable tumor-associated transcript, compared with patients who were CTC negative. Median PFS time was 66.0 days [95% confidence interval (CI) 44.8–87.2] for patients with baseline CTC positivity and 138.0 days (95% CI 124.1–151.9) for CTC-negative patients (p = 0.01, log-rank test). Conclusion: Our results suggest that in addition to the current prognostic methods, CTC analysis represents a potential complementary tool for prediction of outcome in pancreatic cancer patients.

Prognostic and predictive value of circulating tumor cell analysis in colorectal cancer patients.

ObjectiveThe aim of this study was to assess the prognostic and predictive values of circulating tumor cell (CTC) analysis in colorectal cancer patients.Patients and methodsPresence of CTCs was evaluated in 60 colorectal cancer patients before systemic therapy - from which 33 patients were also evaluable for CTC analysis during the first 3 months of treatment - through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 (targeting mucin 1 and EpCAM, respectively), followed by real-time RT-PCR analysis of the tumor-associated genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5.ResultsPatients were stratified into groups according to CTC detection (CTC negative, when all marker genes were negative; and CTC positive when at least one of the marker genes was positive). Patients with CTC positivity at baseline had a significant shorter median progression-free survival (median PFS 181.0 days; 95% CI 146.9-215.1) compared with patients with no CTCs (median PFS 329.0 days; 95% CI 299.6-358.4; Log-rank P < .0001). Moreover, a statistically significant correlation was also founded between CTC detection during treatment and radiographic findings at the 6 month staging. This correlation applied to CTC results before therapy (odds ratio (OR), 6.22), 1 to 4 weeks after beginning of treatment (OR, 5.50), 5 to 8 weeks after beginning of treatment (OR, 7.94) 9 to 12 weeks after beginning of treatment (OR, 14.00) and overall CTC fluctuation during the course of treatment (OR, 20.57).ConclusionThe present study provides evidence of a strong correlation between CTC detection and radiographic disease progression in patients receiving chemotherapy for colorectal cancer. Our results suggest that in addition to the current prognostic factors, CTC analysis represent a potential complementary tool for prediction of colorectal cancer patients’ outcome. Moreover, the present test allows for molecular characterization of CTCs, which may be of relevance to the creation of personalized therapies.

Pretargeting of human mammary carcinoma xenografts with bispecific anti-MUC1/anti-Ga chelate antibodies and immunoscintigraphy with PET

We recently demonstrated the feasibility of combining enhanced tumor-to-tissue contrast and PET imaging for immunoscintigraphic tumor localization in pancreas and colon carcinoma bearing nude mice. Contrast enhancement was obtained with a multistep targeting technique that consists of the sequential administration of an antitumor/antihapten bispecific antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that remains in circulation, and a low molecular weight Ga chelate, labeled with the positron emitter 68Ga, which serves as the hapten. To evaluate the efficacy of this pretargeting technique for breast cancer localization, we synthesized a BS-MAb from the F(ab)(2) fragments of the anti-MUC1 MAb 12H12 which reacts with the vast majority of human breast carcinomas, and the F(ab) fragment of an anti-Ga chelate MAb using a bifunctional chemical linker. The BS-MAb was tested for its affinity and its biokinetics in nude mice bearing a human mammary carcinoma. Equilibrium binding of the BS-MAb for mammary carcinoma cells was low (1.2 x 10(7) M(-1)) while the binding capacity of cells was high (8.4 x 10(6) BS-MAbs per cell). Tumor uptake of the 67Ga labeled chelate in pretargeted animals was to 5.8 +/- 0.8% iD/g resulting in a tumor-to-blood ratio of 2.6 at 1h postinjection. This compares with a ratio of 0.65 and 0.85 obtained with 125I-labeled native 12H12 at 24h and 48h postinjection. No difference in the tumor uptake of both the 68Ga and 67Ga labeled chelate was observed. PET imaging of mice, started 1h postinjection of the 68Ga chelate, clearly visualized all tumors.

Multimarker Analysis of Circulating Tumor Cells in Peripheral Blood of Metastatic Breast Cancer Patients: A Step Forward in Personalized Medicine

Aim: To develop an immunomagnetic assay for the isolation of circulating tumor cells (CTCs) followed by the analysis of a multimarker panel, which will enable the characterization of these malignant cells with high accuracy. Patients and Methods: Peripheral blood (PB) was collected from 32 metastatic breast cancer patients and 42 negative controls. The antibodies BM7 and VU1D9 were used for immunomagnetic tumor cell enrichment. A real-time reverse transcription-polymerase chain reaction (RT-PCR) approach for the markers KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 and ERBB2 was used for CTC detection and characterization. Results: The positivity rates for each marker were as follows: 46.9% for KRT19, 25.0% for SCGB2A2, 28.1% for MUC1, 28.1% for EPCAM, 21.9% for BIRC5, and 15.6% for ERBB2. After the creation of individualized cutoffs, the sensitivity and specificity of the combined marker gene panel increased to 56.3% and 100%, respectively. Interestingly, 27.0% of the HER2-negative tumor patients showed ERBB2 mRNA-positive CTCs. Conclusions: The described technique can be used to measure CTCs with great accuracy. The use of a multimarker panel for the characterization of CTCs may provide real-time information and be of great value in therapy monitoring.

Sensitive detection of micrometastases in bone marrow from patients with breast cancer using immunomagnetic isolation of tumor cells in combination with reverse transcriptase/polymerase chain reaction for cytokeratin-19.

Abstract 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/107–108 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 β2-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 non-malignant cells. For the early diagnosis of disseminating disease, this assay is more efficient than RT-PCR alone and routine immunocytochemistry.

Analysis of sensitivity and specificity of cytokeratin 19 reverse transcriptase/polymerase chain reaction for detection of occult breast cancer in bone marrow and leukapheresis products

Purpose: 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. Materials and methods: 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. Results: 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°C, allowed the detection of one tumour cell in 107 mononuclear cells. None of the control samples (96 peripheral blood and 8 bone marrow) that were positive for β2-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. Conclusion: RT-PCR using CK19-specific primers and optimal experimental conditions is a reliable and specific method for the detection of micrometastatic breast cancer cells.

Evaluating GA733-2 mRNA as a marker for the detection of micrometastatic breast cancer in peripheral blood and bone marrow

Abstract The GA733-2 gene encodes the epithelial glycoprotein 40, a homophilic cell-cell adhesion molecule, which is expressed on the surface of epithelial cells and associated with a variety of carcinomas, e.g. breast, colorectal and lung carcinomas. To test if it could serve as a tumor marker, we have analysed the expression of GA733-2 in bone marrow (BM) and peripheral blood (PB) from healthy donors, and of patients with haematological malignancies or breast cancer using reverse transcriptase-polymerase chain reaction (RT-PCR). The GA733-2 nested PCR was positive in 100% (8/8) of BM and 40% (16/40) of PB from healthy donors, in 100% (33/33) of BM from patients with breast cancer who had no evidence of distant metastases and also in BM and PB from patients with haematological malignancies. GA733-2 mRNA is not specific as a marker for the detection of breast cancer cells in BM and PB.

Prognostic Role of a Multimarker Analysis of Circulating Tumor Cells in Advanced Gastric and Gastroesophageal Adenocarcinomas

Objective: We aimed to assess the prognostic value of circulating tumor cells (CTC) in patients with advanced gastric and gastroesophageal adenocarcinomas. Methods: The presence of CTC was evaluated in 62 patients with advanced gastric and gastroesophageal adenocarcinomas before systemic therapy and at follow-up through immunomagnetic enrichment for mucin 1- and epithelial cell adhesion molecule (EpCAM)-positive cells, followed by real-time RT-PCR of the tumor-associated genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. Results: The patients were stratified into groups according to CTC detection (CTC negative: with all marker genes negative; CTC positive: with at least 1 of the marker genes positive). Patients who were CTC positive at baseline had a significantly shorter median progression-free survival (PFS; 3.5 months, 95% CI: 2.9-4.2) and overall survival (OS; 5.8 months, 95% CI: 4.5-7.0) than patients lacking CTC (PFS 10.7 months, 95% CI: 6.9-14.4, p < 0.001; OS 13.3 months, 95% CI: 8.0-18.6, p = 0.003). Alterations in the marker profile during the course of chemotherapy were not predictive of clinical outcome or response to therapy. Yet, a favorable clinical response depended significantly on CTC negativity (p = 0.03). Conclusion: Our data suggest that the presence of CTC is a major predictor of outcome in patients with gastric and gastroesophageal malignancies.

Evaluation of MUC1 and EGP40 in bone marrow and peripheral blood as a marker for occult breast cancer

Abstractu2002Mucin1 (MUC1) is a class of high molecular weight glycoproteins found in the cell membranes of human epithelial cells. Epithelial glycoprotein 40 (EGP40) is a homophilic cell-cell adhesion molecule and expressed on the surface of most simple epithelial cells and the majority of carcinomas. We analyzed the expression of MUC1 and EGP40 in human bone marrow (BM) and peripheral blood (PB) by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC). Eight BM and 95 PB samples from healthy donors, 39 BM and 17 PB samples from patients with haematological malignancies and 45 BM samples from patients with breast cancer were analyzed. MUC1 mRNA and EGP40 mRNA and protein were detected in BM samples and PB samples from healthy donors and from patients with multiple myeloma (MM), non-Hodgkin’s lymphoma (NHL) and chronic myelogenous leukaemia (CML). The positive cells showed erythroblast-like and plasmacell-like morphology by immunocytochemistry. The MUC1 and EGP40 nested PCR were positive in 83.3% (10/12) and in 100% (33/33) respectively of BM from patiens with breast cancer who had no evidence of distant metastases. It is concluded that MUC1 and EGP40 is expressed in haematopoietic tissues and haematological malignancies. Therefore, MUC1 and EGP40 expression as a marker for detection of breast cancer cells and micrometastatic epithelial cells in BM and PB is not specific using RT-PCR technique and immunocytochemistry.

Evaluation of the reverse transcriptase/polymerase chain reaction for carcinoembryonic antigen for the detection of breast cancer dissemination in bone marrow and peripheral blood

Abstract This study evaluated the specificity and sensitivity of the reverse transcriptase/polymerase chain reaction (RT-PCR) for carcinoembryonic antigen (CEA) for identification of breast tumour cells in bone marrow and in peripheral blood. Using one primer set from the A2/B3 domains of CEA with five to seven mismatches to other CEA-family members allowed reproducible detection of 1 colon tumour cell and 10 breast tumour cells in 107 mononuclear cells. Bone marrow samples from 181 patients with breast cancer were analysed by CEA-RT-PCR; 50 of these samples were analysed in parallel by routine immunocytochemistry. CEA-mRNA-positive bone marrow cells were found in 27.6% of the patients (50/181) with breast cancer. Five immunocytochemistry-positive samples were negative when analysed by CEA-RT-PCR. Limiting factors in the detection of micrometastatic breast tumour cells by CEA-RT-PCR are the heterogeneity of the tumour cells and the deficient expression of CEA in some of these cells. However, CEA-RT-PCR using the specific primer could detect 1 colon tumour cell in 1u2009×u2009107 normal peripheral blood mononuclear cells.