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CHEMOSENSITIVITY TESTING METHODOLOGY

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IN ADVANCE CASE OF DISEASE (STAGE IIIb AND iv) THE SAMPLE CAN BE A WHOLE BLOOD ... SEEKED FOR SIMPLE TEST FOR PREDICTION OF TREATMENT OPTION FOR EACH PATIENT. ... – PowerPoint PPT presentation

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Title: CHEMOSENSITIVITY TESTING METHODOLOGY


1
CHEMOSENSITIVITY TESTING METHODOLOGY
  • ISOLATION OF FROM SAMPLE CANCER CELLS AND
    DEVELOPMENT OF PRIMARY CULTURES.
  • EACH CULTURE INCLUDES IN THE CULTIVATION MEDIA
    A CYTOSTATIC DRUG.
  • TESTING OF GENES BY MICRO-ARRAY ANALYSIS
    THAT BECOME TARGETS FOR CYTOSTATICS, OR
    THEY INVOLVED IN RESISTANCE PHENOTYPE OR THEY
    INVOLVED TO THE METASTATIC PROCEDURE.
  • VERIFICATION OF THE ANALYSIS BY SMALL SCALE
    VIABILITY TEST OF THE CULTURES.

R.G.C.C.-Research Genetic Cancer Centre-Web site
www.rgcc-genlab.com
2
ISOLATION OF CANCER CELLS FROM A SAMPLE
  • IN ADVANCE CASE OF DISEASE (STAGE IIIb AND
    iv) THE SAMPLE CAN BE A WHOLE BLOOD SAMPLE
    IN MINIMUM AMOUNT OFG 15ML (CIRCULATING TUMOUR
    CELLS).
  • From the blood sample using
    monoclonal antibody anti-CD45 we track every
    blood origin cell and using after magnetic
    field all blood origin cells are
    concentrate to the bottom of the vial. The
    upper layer of the vial contains the
    cancer cells.
  • After discard the pellet we use a
    monoclonal antibody against a specific
    membrane marker for cancer cells. Then using
    again magnetic field the cancer cells are
    collected from the bottom of the vial.
  • IN EARLIER STAGE THE SAMPLE MUST BE TISSUE
    SAMPLE FOR AVOIDING THE INCIDENT NOT TO
    FIND CANCER CELLS TO THE THE BLOOD SAMPLE.

R.G.C.C.-Research Genetic Cancer Centre-Web site
www.rgcc-genlab.com
3
MICRO-ARRAY ANALYSIS VERIFICATION
  • THE METHODOLODY OF MICRO-ARRAY
  • ISOLATION OF mRNA FROM CANCER CELLS
  • PRODUCTION OF cDNA FROM mRNA USING RT-PCR
    (REVERSE TRANSCRIPTATION PCR).
  • MODIFICATION OF cDNA WITH BIOTIN-STREPTAVIDIN
  • HYBRIDIZATION OF cDNA WITH SPECIFIC PROBES FOR
    GENES
  • VISUALISATION OF HYBRIDIZED PROBES
  • GENES THAT ANALYSED
  • GENES THAT BECOME TARGETS FOR CYTOSTATICS
    (DHFR,TS, TOPOI, TOPOII etc)
  • GENES THAT THEY PRODUCED GROWTH FACTORS OR
    GROWTH FACTORS RECEPTOR (EGF, IGF , TGF etc)
  • GENES THAT THEY INVOLVED TO METASTASES (VEGF,
    EGF, PDGF ETC)
  • GENES THAT REGULATES THE CELL CYCLE (p53, p27,
    p16 etc)
  • GENES THAT THEY INVOLVED TO RESISTANCE PHENOTYPE
    (MDR1, DNMT1,BCL, NP, NFK, HTERT etc)
  • VERIFICATION OF THE RESULTS BY VIABILITY TEST
    USING TRYPAN BLUE 0.4

R.G.C.C.-Research Genetic Cancer Centre-Web site
www.rgcc-genlab.com
4
ADVANTAGES OF MICRO-ARRAY VERSUS PREVIOUS
TECHNIQUES USED IN CHEMOSENSITIVITY TESTING
  • FROM PAST TIMES ONCOLOGY
    SEEKED FOR SIMPLE TEST FOR PREDICTION OF
    TREATMENT OPTION FOR EACH PATIENT. HENCE
    MANY CHEMOSENSITIVITY TESTING ASSAYS HAVE
    BEEN DEVELOPED IN THE PAST BASED ON SIMPLE
    POINTS AND USING CELLULAR-VIABILITY
    TECHNIQUES.
  • MICRO-ARRAY ANALYSIS PROVIDE
    US THE ABILITY TO TEST THE EXPRESSION OF
    MUTIPLE GENES AT THE SAME TIME.
  • PREVIOUS TECHNIQUES ARE BASED MAINLY IN
    VIABILITY COLORIMETRIC TECHNIQUES AND THEY HAVE
    HIGH RATE OF FALSE POSITIVE AND FALSE
    NEGATIVE RESULTS . THOSE INSIDENTS ARE REDUCED
    DRAMATICALLY BY MICRO-ARRAY ANALYSIS
  • PREVIOUS TECHNIQUES NEEDS GREAT SPENDING OF
    TIME TO COMPLETE THE ANALYSIS (40 DAYS AND
    EVEN MORE). MICRO-ARRAY ANALYSIS NEED ONLY
    6-7 DAYS
  • PREVIOUS TECHNIQUES NEED ONLY TISSUE SAMPLES
    WHICH NOT ALWAYS AVAILIABLE, ESPECIALLY IN
    ADVANCE STAGE OF DISEASE ON PATIENT WHICH
    ARE MULTITREATED. MICRO-ARRAY CAN BE MADE BY
    ISOLATION OF CIRCULATING CANCER CELLS.
  • PREVIOUS TECHNIQUES CANNOT PROVIDE TO
    CLINICIAN MANY DETAILS ABOUT THE POTENTIALS
    OF CANCER FOR A SPECIEFIC PATIENT .
    MICRO-ARRAY CAN PROVIDE INFORMATIONS
    CONCERNING METASTATIC ABILITY OF THE TUMOUR
    , THE RESISTANCE MECHANISM AND HOW CAN BE
    REVERSED etc
  • PREVIOUS TECHNIQUES USING CELL CULTURE
    DEVELPOPMENT AND THEY TEST A CELL LINE FROM
    TISSUE SAMPLE. BUT CELL LINE IS ONLY ONE
    SUBCLONE FROM THE TOTAL METASTATIC CANCER
    CELL POPULATION. THEREFOR THE ANALYSIS
    PROVIDE INFORMATION ONLY FOR THIS SUBCLONE.
    IN MICRO-ARRAY THE CULTIVATION IS IN SHORT
    PERIOD (PRIMATY CULTURES) TO AVOID THE
    DISCLOSURE OF MANY SUBCLONES . HENCE THE
    ANALYSIS INVOLVES ALL THE METASTATIC
    SUBCLONES FROM THE TUMOUR.
  • IN FEW WORDS MICRO-ARRAY ANALYSIS OFFERS
  • ACCURACY, SPECIFICITY, SPEED
  • ALL THOSE MAKES MICRO-ARRAY AN INOVATIVE
    POWERFUL TOOL TO THE HANDS OF ONCOLOGY
    THERAPIST (SURGEON OR INTERNIST)

R.G.C.C.-Research Genetic Cancer Centre-Web site
www.rgcc-genlab.com
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