Sensitive detection of rare cancer cells in sputum and peripheral blood samples of patients with lung cancer by preproGRP-specific RT-PCR

Abstract Studies have shown that primary tumor sites begin shedding cancerous cells into peripheral blood at early stages of cancer, and the presence and frequency of circulating tumor cells (CTCs) in blood is directly proportional to disease progression. The challenge is that the concentration of the CTCs in peripheral blood may be extremely low. In the past few years, several microfluidic-based concepts have been investigated to isolate CTCs from whole blood. However, these devices are generally hampered by complex fabrication processes and very low volumetric throughputs, which may not be practical for rapid clinical applications. This paper presents a high-performance yet simple magnetophoretic microfluidic chip for the enrichment and on-chip analysis of rare CTCs from blood. Microscopic and flow cytometric assays developed for selection of cancer cell lines, selection of monoclonal antibodies, and optimization of bead coupling are discussed. Additionally, on-chip characterization of rare cancer cells using high resolution immunofluorescence microscopy and modeling results for prediction of CTC capture length are presented. The device has the ability to interface directly with on-chip pre and post processing modules such as mixing, incubation, and automated image analysis systems. These features will enable us to isolate rare cancer cells from whole blood and detect them on the chip with subcellular resolution.

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Quantitative detection of circulating renal cancer cells in peripheral blood samples: Experience of a two-center study

European Urology Supplements ◽

10.1016/s1569-9056(03)80709-1 ◽

2003 ◽

Vol 2 (1) ◽

pp. 179

Author(s):

A. Meye ◽

U. Bilkenroth ◽

U. Schmidt ◽

Ms. Blümke ◽

S. Füssel ◽

...

Keyword(s):

Cancer Cells ◽

Renal Cancer ◽

Peripheral Blood ◽

Quantitative Detection ◽

Blood Samples ◽

Center Study

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Prognostic Value of Circulating Melanoma Cells Detected by Reverse Transcriptase–Polymerase Chain Reaction

Journal of Clinical Oncology ◽

10.1200/jco.2003.01.128 ◽

2003 ◽

Vol 21 (5) ◽

pp. 767-773 ◽

Cited By ~ 65

Author(s):

Giuseppe Palmieri ◽

Paolo A. Ascierto ◽

Francesco Perrone ◽

Sabrina M.R. Satriano ◽

Alessandro Ottaiano ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Subgroup Analysis ◽

Peripheral Blood ◽

Predictive Value ◽

Prognostic Value ◽

Rt Pcr ◽

Chain Reaction ◽

Blood Samples ◽

Stage Of Disease ◽

Polymerase Chain

Purpose: Factors that are predictive of prognosis in patients who are diagnosed with malignant melanoma (MM) are widely awaited. Detection of circulating melanoma cells (CMCs) by reverse transcriptase-polymerase chain reaction (RT-PCR) has recently been postulated as a possible negative prognostic factor. Two main questions were addressed: first, whether the presence of CMCs, defined as the patient being positive for any of the three markers, had a prognostic role; and second, what the predictive value of each individual marker was. Patients and Methods: A consecutive series of 200 melanoma patients observed between January 1997 and December 1997, with stage of disease ranging from I to IV, was analyzed by semiquantitative RT-PCR. Tyrosinase, p97, and MelanA/MART1 were used as markers to CMCs on baseline peripheral blood samples. Progression-free survival (PFS) was used as a unique end point and was described by the product limit method. Multivariable analysis was applied to verify whether the auspicated prognostic value of these markers was independent of the stage of disease, and a subgroup analysis was performed that excluded patients with stage IV disease. Results: Overall, 32% (64 of 200) of patients progressed, and a median PFS of 52 months in the whole series was observed. The presence of CMCs and the markers individually or combined was predictive of prognosis in the univariate analysis but did not provide additional prognostic information to the stage of disease in multivariable models. In the subgroup analysis of stage (ie, I–III subgroup), similar results were observed. Conclusion: Detection of CMCs in peripheral blood samples at the time of MM diagnosis by semiquantitative RT-PCR does not add any significant predictive value to the stage of disease. Thus, this approach should not be used in clinical practice, and further studies are required to determine its usefulness.

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Simultaneous sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level

Chemical Science ◽

10.1039/c7sc04296e ◽

2018 ◽

Vol 9 (3) ◽

pp. 712-720 ◽

Cited By ~ 36

Author(s):

Juan Hu ◽

Ming-hao Liu ◽

Ying Li ◽

Bo Tang ◽

Chun-yang Zhang

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Single Molecule ◽

Simultaneous Detection ◽

Sensitive Detection ◽

Lung Cancer Cells ◽

Dna Glycosylase ◽

Dna Glycosylases ◽

Single Molecule Level ◽

Alkyladenine Dna Glycosylase

We demonstrate the simultaneous detection of human 8-oxoguanine DNA glycosylase 1 and human alkyladenine DNA glycosylase at the single-molecule level.

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Partial Tandem Duplications of the MLL Gene Are Detectable in Peripheral Blood and Bone Marrow of Nearly All Healthy Donors

Blood ◽

10.1182/blood.v92.5.1728.417a01_1728_1734 ◽

1998 ◽

Vol 92 (5) ◽

pp. 1728-1734 ◽

Cited By ~ 1

Author(s):

Susanne Schnittger ◽

Bernhard Wörmann ◽

Wolfgang Hiddemann ◽

Frank Griesinger

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Chromosome 11 ◽

Rt Pcr ◽

Blood Samples ◽

Mll Gene ◽

Oncogenic Mutations ◽

Healthy Donors ◽

American Society

Partial tandem duplication within the MLL gene has recently been described as a novel genetic alteration in acute myeloid leukemia (AML). It has been associated with trisomy of chromosome 11, but was also identified in AML patients with normal karyotypes. The current study was performed to investigate whether MLL duplications are restricted to AML, and hence whether they may also occur in normal hematopoietic cells. MLL-duplication transcripts were analyzed by nested reverse-transcriptase polymerase chain reaction (RT-PCR) in peripheral blood in two groups of 45 and 20 patients, respectively, as well as in two bone marrow samples from healthy volunteers. Duplications were detected in two independent nested RT-PCR experiments in the peripheral blood samples of 38 of 45 (84%) and 20 of 20 (100%) of the two groups and in both bone marrow samples. On this basis, MLL duplications seem to occur frequently in a subset of cells in normal hematopoiesis. The type of partially duplicated MLL transcripts varied substantially. Three transcripts were identical to those known from AML. In addition, four new transcripts were characterized. Three of these four were in frame and potentially translatable. MLL duplications were also detected by seminested genomic PCR with intron 9– and intron 1–specific primers in 20 of 20 peripheral blood samples studied, indicating that the duplications are genomically fixed at the DNA level and are not an RT-PCR artifact. In summary, MLL duplications are regularly generated by homologous ALU recombination in a small number of hematopoietic cells of most or even all healthy donors. These data suggest that MLL duplications are not implicated in the malignant transformation in AML, or alternatively, that only a few cells will acquire additional oncogenic mutations necessary to establish the malignant phenotype of AML. © 1998 by The American Society of Hematology.

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Microarray study of gene expression profiles in peripheral blood samples from lung cancer patients before and after erlotinib treatment

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e19072 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e19072-e19072

Author(s):

A. Irigoyen ◽

C. Olmedo ◽

J. Valdivia ◽

A. Comino ◽

C. Cano ◽

...

Keyword(s):

Gene Expression ◽

Lung Cancer ◽

Growth Factor ◽

Healthy Volunteers ◽

Cancer Patients ◽

Peripheral Blood ◽

Expression Profiles ◽

Control Group ◽

Blood Samples ◽

Lung Cancer Patients

e19072 Background: The gene expression profile in peripheral blood samples from lung cancer patients is a potential predictor to treatment response. Methods: The study has been developed using 10 healthy volunteers as the control group and 10 lung cancer patients (stage IV). Written informed consent was obtained being the protocol approved by the local Clinical Research and Ethics Committee. Peripheral blood samples were obtained from lung cancer patients before (T0) and after treatment (T15d). RNA from peripheral blood samples was extracted and purified selecting 28S/18S ratios>1.5 to obtain cDNA and cRNA for hybridization of the 20,000 genes included in Human 20K CodeLink. An array from each participant was obtained in duplicate. For each array, 2 μg of cRNA was compared to 2 μg of healthy cRNA.. Significant genes were found using Significance Analysis of Microarrays which uses repeated permutations of the data. Results: The selected genes were expressed >3-fold with a false discovery rate =0.05. Before treatment (T0) when patients were compared to healthy volunteers there was an increase in the expression of: histone 1 H4c, transforming growth factor beta 2, endothelial cell growth factor 1 (platelet-derived), glucose-6-phosphatase catalytic 2, Relaxin 3 receptor 1, Insulin-like growth factor binding protein 2, RAS-like family 11 member B, and ELK4. After treatment (T15d), when each lung cancer patient's results were compared to their own before treatment results (T0), there was an increase in the expression of: Bcl2, myosin light polypeptide 4; interferon alpha-inducible protein 27; interferon gamma receptor 1; RASSF5, ARHGEF6, IGFBP5, tumor protein p53 inducible nuclear protein 1, peroxisome proliferative activated receptor gamma. Conclusions: The data presented identifies biologically relevant over-expressed genes in lung cancer. A validation of these results and the analysis of the genes that identify patients who will respond positively to erlotinib treatment is being carried out. No significant financial relationships to disclose.

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Impact of Molecular Staging Methods in Primary Melanoma: Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) of Ultrasound-Guided Aspirate of the Sentinel Node Does Not Improve Diagnostic Accuracy, But RT-PCR of Peripheral Blood Does Predict Survival

Journal of Clinical Oncology ◽

10.1200/jco.2007.13.7653 ◽

2008 ◽

Vol 26 (35) ◽

pp. 5742-5747 ◽

Cited By ~ 11

Author(s):

Christiane A. Voit ◽

Gregor Schäfer-Hesterberg ◽

Martina Kron ◽

Alexander C.J. van Akkooi ◽

Juergen Rademaker ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcriptase ◽

Peripheral Blood ◽

Primary Melanoma ◽

Disease Free Survival ◽

Rt Pcr ◽

Ultrasound Guided ◽

Chain Reaction ◽

Blood Samples ◽

Polymerase Chain

Purpose This study analyzes (1) the value of tyrosinase reverse-transcriptase polymerase chain reaction (RT-PCR) of aspirates obtained by ultrasound-guided fine-needle aspiration cytology (US-FNAC) of sentinel nodes (SNs) in patients with melanoma before sentinel lymph node biopsy (SLNB) and (2) the value of RT-PCR of blood samples of all SLNB patients. Patients and Methods Between 2001 and 2003, 127 patients with melanoma (median Breslow depth, 2.1 mm) underwent SLNB. FNAC was performed in all SNs of all patients pre- and post-SLNB. The aspirates were partly shock-frozen for RT-PCR and were partly used for standard cytology. Peripheral blood was collected at the time of SLNB and at every outpatient visit thereafter. Results Thirty-four (23%) of 120 SNs were positive for melanoma. SN involvement was predicted by US-FNAC with a sensitivity of 82% and a specificity of 72%. Additional tyrosinase RT-PCR revealed the same sensitivity of 82% and a specificity of 72%. At a median follow-up time of 40 months from first blood sample, peripheral-blood RT-PCR was a significant independent predictor of disease-free survival (DFS) and overall survival (OS; P < .001). Conclusion US-FNAC is highly accurate and eliminates the need for SLNB in 16% of all SLNB patients. RT-PCR of the aspirate or excised SN does not improve sensitivity or specificity. RT-PCR of blood samples predicts DFS and OS.

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The value of SHOX2 methylation test in peripheral blood samples used for the differential diagnosis of lung cancer and other lung disorders

Neoplasma ◽

10.4149/210_150419n208 ◽

2016 ◽

Cited By ~ 3

Author(s):

M. KONECNY ◽

J. MARKUS ◽

I. WACZULIKOVA ◽

L. DOLESOVA ◽

R. KOZLOVA ◽

...

Keyword(s):

Lung Cancer ◽

Differential Diagnosis ◽

Peripheral Blood ◽

Blood Samples ◽

Diagnosis Of Lung Cancer

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Low specificity of cytokeratin 19 reverse transcriptase-polymerase chain reaction analyses for detection of hematogenous lung cancer dissemination.

Journal of Clinical Oncology ◽

10.1200/jco.1995.13.11.2769 ◽

1995 ◽

Vol 13 (11) ◽

pp. 2769-2775 ◽

Cited By ~ 125

Author(s):

M Krismann ◽

B Todt ◽

J Schröder ◽

D Gareis ◽

K M Müller ◽

...

Keyword(s):

Lung Cancer ◽

Cancer Patients ◽

Peripheral Blood ◽

Lung Tumor ◽

Cytokeratin 19 ◽

Healthy Controls ◽

Rt Pcr ◽

Lung Cancer Patients ◽

Tumor Dissemination ◽

Breast And Prostate Cancer

PURPOSE Sensitive detection of systemic tumor dissemination in lung cancer patients is important for selection of appropriate treatment modalities. Based on recent promising data that showed reverse transcriptase-polymerase chain reaction (RT-PCR) analyses for cytokeratin 19 (CK-19) expression in peripheral-blood or bone marrow samples to be a rapid and highly sensitive method for detection of hematogenous tumor dissemination in patients with breast and prostate cancer, we evaluated the specificity of this assay system in lung cancer patients and a large number of healthy controls. PATIENTS AND METHODS We examined CK-19 mRNA expression by RT-PCR in 17 lung cancer cell lines and in peripheral-blood samples of 50 lung tumor patients and 65 healthy controls. RESULTS Expression of CK-19 mRNA was observed in all lung cancer cell lines and in 50% of peripheral-blood samples from lung tumor patients. However, under the experimental conditions analyzed, at least 20% of the control samples were positive for CK-19 mRNA expression. CONCLUSION Contrary to prior reports, RT-PCR may detect non-tissue-specific constitutive low-level (illegitimate) expression of CK-19 mRNA in peripheral-blood mononuclear (PBMN) cells in a significant number of healthy controls. This finding may not only hamper the use of this assay system in lung cancer patients, but also questions its proposed applicability in patients with other epithelial tumors such as breast and prostate cancer.

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