Microsatellite Instability: Correlation with Chemotherapeutic Drugs

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Microsatellite InstabilityCorrelation with
Chemotherapeutic Drugs

• Cheryl Bline
• Dr Elisabeth Arévalo
• Providence College

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Purpose

• Examine the incidence of microsatellite
  instability in colorectal cancer for the
  diagnosis and determination of the response to
  chemotherapeutic drugs

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Microsatellites

• Tandem repeats of two to four bases

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Instability

• Number of repeats strictly regulated in healthy
  individuals
• Cancer patients show instability
• Either an increase or a decrease in the number of
  repeats

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Mismatch Repair Proteins

• Corrects mismatched nucleotides inserted during
  DNA replication
• Instability leads to nonfunctioning mismatch
  repair proteins
• Nonfunctioning proteins cannot correct mistakes
  in DNA replication

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Tumor Suppressor

• Prevent cells from becoming cancerous
• Expect instability in mismatch repair genes will
  correlate with mutations in tumor suppressor
  genes
• Lead to nonfunctioning tumor suppressor gene

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Isolated Pelvic Perfusion

• Isolate the pelvic cavity through tourniquets on
  the thighs and balloons in the aorta and the
  inferior vena cava
• Chemotherapeutic cocktail is infused into the
  pelvic cavity

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Isolated Pelvic Perfusion
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Procedure

• Procedure lasts for 1 hour
• Drugs are infused during first 25 minutes
• Blood samples taken at five minute intervals
  throughout procedure
• Systemic
• Pelvic
• Biopsy sample obtained when accessible

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Chemotherapeutic Cocktail
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Samples
Number of Patients
8 Control Samples
RCC Recurrent colorectal cancer ROC Recurrent
ovarian cancer PS Pelvic Sarcoma
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Samples

• Samples from the pelvic and systemic were taken
  from early and late times in the procedure
  (usually 10 and 60 minutes)
• Plasma samples used for patients when blood not
  available
• Tissue samples were also used when available

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Methods

• Centrifuge blood samples to separate plasma
• Extract DNA from blood using QIAamp DNA Blood
  Mini Kit (81 samples)
• PCR performed and product labeled with 35S
• Gel electrophoresis performed to separate samples
  by size
• Samples visualized by autoradiography

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Analysis

• PCR primers
• 11 loci for mismatch repair genes
• 4 loci for cell growth pathways
• Locus- position of a gene on a chromosome
• Allele- one set of alternative forms of a gene
• Polyacrylamide gels were ran to determine the
  amount of allelic variation and to assess the
  degree of instability from these results

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Analysis

• Control samples (8) from individuals who are not
  known to have cancer were used for comparison
• Samples were also compared between pelvic and
  systemic within one patient
• The samples were ran against a known ladder
  sequence (m13mp18) to determine size

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Results

• Differences within one patient
• D5S346
• D17S250
• Differences between patients and control
• ACTC
• D3S1611
• Special case

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Fixed Differences Within Patients Samples
Each patient shown with pelvic and systemic
samples, C denotes control samples Allele sizes
determined with the use of m13mp18
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Fixed Differences Between Patient and Control
D3S1611
ACTC
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Special Case

• One patient went into remission for two years
• First procedure in January 2003
• Cisplatin
• Second procedure in March 2003
• Oxaliplatin
• Remission for two years
• Third procedure March 2005

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Conclusions

• Variation
• Within one patient
• Between patients and controls
• Variation may be useful in diagnosis and
  determining the effectiveness of treatment

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Further Research

• Examine differences in patient that attained
  remission
• How did the alleles change over time
• How do the alleles compare to other patients who
  do not attain remission
• Sequence with the cell growth genes
• TGF-?-RII, ACTC, TP53, BAX

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Acknowledgement

• Dr James Belliveau (PC Chemistry Dept)
• Dr Harold Wanebo and Roger Williams Medical
  Center
• PC Students
• Carol Geddes (04), Nate Mockler (06), Jen
  Deiana (05), Elizabeth Benz (07), Julie
  Mausolf-Mark (07)

(Supported by BRIN/INBRE from the NIH)
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References

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