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					                                                                            Isolation, Detection + Cellular and Molecular Characterization of
                                                                                    Circulating Tumor Cells in Breast Cancer Patients
  Harlem Children                                                                                                                        Pankaj K. Saha, Dr. Satyajit Bhattacharya Ph D.
  Society                                                                                                                      Minimal Residual Lab, Memorial Sloan-Kettering Cancer Center, New York City, NY, 10021, U.S.A

 Abstract:                                                                                                                                                                                                                                                                Pictures (Immunomagneic Method):
                                                                                                                                           Materials and Methods:
 It is my hypothesis that circulating breast cells can be detected, isolated and characterized from breast cancer patients. Six highly
 progressive metastatic breast cancer patients were analyzed for this study. The breast cells were isolated by a Novel sensitive           1. Specimen collection:
 Immunomagnetic Method from the mononuclear fraction isolated from leukapheresis. Nested RT-PCR for Mammoglobin was
 performed. Breast cells were then used for microscopic evaluation, and were enriched immuno-magnetically and cytospun. Slides             Highly progressive metastatic breast cancer patients were analyzed for the study. All the patients were positive for
 processed by immunoperoxidase (IPX) were evaluated for the detection of Mammoglobin. Other slides were stained with propidium             metastases by imaging, bone scan, and CT scan. A group of healthy individuals with no apparent malignancies were taken as
 Iodide (PI, DNA Content red) and subjected to immunofluorescence for Mammoglobin (green) and analyzed by a laser Scanning                 negative controls. Informed consent from all the patients prior to participation in the study was obtained. The peripheral
 Microscope Cytometer.                                                                                                                     blood was mixed with appropriate amount of phosphate buffered saline (PBS), and separated using Ficoll gradient by density
                                                                                                                                           gradient centrifugation. The mononuclear and a portion of the granulocytic layer were taken for further study.
 Results: Detectable levels of Mammoglobin mRNA was observed in 3 out of every 6 patients, and detectable levels of Actin was
 observed in 6 out of every 6 patients.                                                                                                    2. Epithelial Cell Enrichment:

 Conclusion: We have been able to isolate circulating breast cells using a novel immunobead technique for microscopic evaluation by        One half of the mononuclear cell fraction isolated from the blood samples was incubated with magnetic beads coated with
 IPX, LSMC, and RT-PCR. Use of multiple markers greatly enhances the Molecular detection of circulating breast cells. Mammoglobin          Ber-EP4 antibody directed against human epithelial antigen, a membrane antigen widely expressed in epithelial cells. The
 seems to play a critical role in the proliferation in breast cancer. This technique can be used for Molecular Characterization of the     other half of the mononuclear cell fraction (used for microscopic evaluation) was incubated with magnetic beads coated with
 CTC cells in Breast and other solid tumors. This would help Assess, Stratify, and Design Targeted Therapy for Cancer Patients .           Ber-EP4 antibody linked to the magnetic beads via a DNA linker. The magnetic cell fraction was isolated and treated with
                                                                                                                                           DNase to remove the magnetic beads from the cells. The isolated Immunomagnetic bead-free cells were then cytospun, fixed
                                                                                                                                           in Methanol: Acetone (1:1), and stored at -80°C.
                                                                                                                                           3. RNA Isolation:

                                                                                                                                           Messenger RNA was isolated using Oligo (dT) attached to magnetic beads after epithelial cell enrichment. The isolated RNA
Introduction:                                                                                                                              was stored at -80°C until further used.

                                                                                                                                           4. RT-PCR for Specific mRNAs and Gel Electrophoreses:
       Cancer is often referred to as “the silent killer.” At the onset of cancer, cancer cells are difficult to
identify; by the time they have been detected, not much can be done to cure the patient. Cancer is                                         Single tube, nested RT-PCR for Mammoglobin mRNA was performed on the RNA samples. The PCR amplified products
characterized as abnormal cells that divide and multiply at an uncontrollable rate. It can be genetically                                  were run on a 2% ethidium bromide agarose gels and observed under a UV transilluminator. To confirm the presence of
                                                                                                                                           amplifiable RNA, every sample was subjected to RT-PCR for actin mRNA, and confirmed on agarose gel electrophoresis.
inherited or may develop through environmental factors. Examples that contribute to environmental
factors are inhalation of toxic fumes such as insecticides & pesticides, extensive exposure to radiation                                   5. Positive and Negative RT-PCR Controls:
(UV Rays from the sun and other sources), the over intake of foods with high lipid content, and the
                                                                                                                                           MCF-7 (Breast cell line) and Carcinoma tissue from Breast Cancer Patients were used as positive controls. Negative control
construction of nuclear power plants.                                                                                                      PCR consisted of reactions mixtures without any template.
       Tumors may develop in any organ of the body. Eventually the cancer cells metastasize and evade
other organs of the body. In general, cancer cells can travel in the body through two ways, the lymphatic                                  6. Sensitivity Experiments:                                                                                                   Results:
                                                                                                                                                                                                                                                                                                                         Immunobead RT-PCR in Breast Cancer Patients
system and the bloodstream. The lymphatic system is the body’s secondary immune system and the                                             MCF-7 (breast cell line) was used to define the sensitivity of the assays. The cell line was grown and maintained in tissue
bloodstream is where red blood cells (RBC) circulate to supply the body with oxygen and nutrients. As                                      culture. The MCF-7 cells were mixed with whole blood samples from healthy volunteers to generate serial dilutions.
cancer progresses the tumor is more visible to the patient because it increases in size. By the time a                                     Subsequently, they were processed for further RNA analysis.

tumor forms into a small lump, there are more than a billion cancer cells there.                                                           7. Immunohistochemistry (IHC) and Immunofluorescence (IMF):
       Treatments should be taken by the patient to prevent the progression of the tumor. Individuals
with cancer may have surgery, where the tumor is physically removed. They may also take                                                    The slides were processed either by IHC of by IMF. Slides processed for IHC were treated with mouse anti human
                                                                                                                                           Mammoglobin, antibody (Hybritech) after pretreatment with Citric acid solution. They were then stained with DAB after
chemotherapy, where anticancer drugs are used for treatment. Biological therapy can be used to                                             treatment with horse anti mouse, IgG and peroxide conjugated with streptavidin. These cells were observed under light
stimulate cells to restore their ability to fight infections and other diseases. Radiation therapy is an                                   microscopy after counter staining with Hematoxilin.
alternative option; It uses high-energy radiation from computerized axial tomography (CAT scan),                                           The other group of slides processed for cell cycle evaluation were treated with Hybritech anti – Mammoglobin antibody and
                                                                                                                                           then with rabbit anti mouse F(ab’)2-FITC. These were then treated with Propidium Iodide (5 ug/ml) in the presence of
magnetic resonance imaging (MRI), gamma rays, neutrons, and other sources to kill cancer cells and                                         RNAseA (100 ug/ml) for 30 minutes at room temperature in the dark. The fluorescence – green (tyrosinase) and red (PI,
shrink tumors. Exposure to extensive amounts radiation may also causes side affects. It suppresses the                                     DNAcontent) was analyzed under a Laser Scanning Microscope Cytometer (LSMC) (Compucyte Corp., Cambridge, MA).
body’s immune system and makes patient more prone to other diseases; patients may even die of
common cold.
                                                                                                                                                                                                                                                                                                              Actin PCR B/BM: 154bp                    Mammoglobin PCR: 215bp
       Treatments do not eliminate all of the cancer cells; they just reduce the number of the cancer cells.                                                                                                                                                                                                  Of the six patients that were            Out of the 6 tested only 3 of
The remaining cells become dormant and go into circulation in the blood; they carry out normal cell
functions. These cancer cells in the blood are known as circulating tumor cells (CTC) and are
                                                                                                                                          Pictures (Guanidium Isothicyanide Method):                                                                                                                          tested, all six had Actin         them had Mammoglobin

characterized by the type of molecular marker they have.
                                                                                                                                                                                                                                                                          (i) Magnetic phase method is more efficient than the Guanidium Isothicyanide Method Using Ultraspec
       Tumor markers are branches in specific cells that can often be detected in higher-than-normal
amounts in the blood of patients with a certain type of cancer. There is a specific molecular marker of                                                                                                                                                                   (ii) Cancer cells can be detected at early stages (treatment and monitoring of the cancer patients can be
every type of cell; it is these markers that help differentiate, characterize, and distinguish one cell from                                                                                                                                                              enhanced and customized)

another. The molecular markers for breast cells are Mammoglobin and cytokeratin-19. Tumor markers                                                                                                                                                                         (iii) Less radiation therapy, chemotherapy, and immunotherapy
are produced by the tumor itself or by the body in response to the presence of cancer or non-cancerous
conditions. Tumor marker levels can be useful in the detection and diagnosis of different types of
cancer.                                                                                                                                                                                                                                                                  Acknowledgments
                                                                                                                                                                                                                                                                         Memorial Sloan-Kettering Cancer Research Center
                                                                                                                                                                                                                                                                         Harlem Children Society
Objectives                                                                                                                                                                                                                                                               Manhattan Center for Science and Mathematics
                                                                                                                                                                                                                                                                         Dr. Sat Bhattacharya
        (i.)   To isolate Circulating breast cells from Peripheral blood of carcinoma patients with breast cancer using a
        Novel Immunobead Technique for microscopic evaluation by Immunohistochemistry and for Laser Scanning                                                                                                                                                             Mrs. Charlene Chan
        Microscope Cytometer by Immunofluorescence.                                                                                                                                                                                                                      Mr. Carlos Franco
        (ii.)  To detect mRNA by Immunobead RT-PCR.

Hypothesis                                                                                                                                                                                                                                                                   Cotran, Kumar, Collins: The Pathological Basis of Disease 6th Edition (W.B. Saunders Company)

Positive selection of epithelial cells using immunobead enrichment of peripheral blood samples from breast cancer patients allows:                                                                                                                                           Foss AJ, Guille MJ, Occleston NL, Hykin PG, Hungerford JL, and LightmanS: The detection of melanoma cells in peripheral blood by
         (i.)     Detection and estimation of breast cancer cells using RT-PCR for Mammoglobin mRNA by                                                                                                                                                                        reverse transcriptase polymerase chain reaction. Br J Cancer 72: 155 – 159, 1995
         Immunohistochemistry (for Light Microscopic examination), and by Immunofluorescence (for Laser Scanning
         Microscopic Cytometer).                                                                                                                                                                                                                                             Ghossein RA, Bhattacharya Coit DG. Reverse transcriptase polymerase chain reaction (RT-PCR) detection of melanoma-related transcripts in
                                                                                                                                                                                                                                                                              the peripheral blood and bone marrow of patients with malignant melanoma. What have we learned? Recent Results Cancer Res.
         (ii.)    Detection of Mammoglobin by RT-PCR                                                                                                                                                                                                                          2001;158:63-77.

Principles                                                                                                                                                                                                                                                                   Ghossein RA, Bhattacharya S.: Molecular detection and characterization of circulating tumour cells and micrometastases in solid tumours.
                                                                                                                                                                                                                                                                              Eur J Cancer. 2000 Aug 36 (13 Spec No):1681-94.

        (i)     Positive Selection of epithelial cells is possible using Immunomagnetic beads coated with Ber-EP4          monoclonal                                                                                                                                        Pelkey TJ, Frierson HF and Bruns DE: Molecular and immunological detection of circulating tumor cells and micrometastases from solid
        antibody directed against the human epithelial antigen, expressed on most normal and neoplastic    human epithelial tissues.                                                                                                                                          tumors. Clin Chem 42:1369 – 1381, 1996
        (ii)    Messenger ribonucleic (mRNA) is very unstable in extracellular environment; therefore, detection or mRNA
                should indicate the presence of tumor cells in the examined tissue of body fluid.                                                                                                                                                                            Ronald A. Ghossien, Leo Carusone, and Satyajit Bhattacharya: Molecular detection of micrometastases and circulating tumor cells in
        (iii)   PolyA + mRNA can be isolated from selected cells using Immunomagnetic beads coated with Oligo (dT)                                                                                                                                                            melanoma prostatic and breast carcinomas. In vivo 14: 237 – 250, 2000