Grand Rounds Breast Cancer Biomarkers

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Grand RoundsBreast Cancer Biomarkers

• Coy Heldermon
• October 14, 2005

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Cases

• 60yo wm presents with urinary hesitancy.
• 50yo wf presents with a colon mass.
• 45yo bf presents with bloating and swollen belly.
• 20yo wm presents with gynecomastia.

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Cases

• What are you going to check?
• What are you going to follow after resection of
  the malignancy that each has?
• Does anything you check tell you the prognosis of
  their disease?
• Does anything you check affect how aggressively
  you treat them?
• Are you going to follow anything to tell you when
  further treatment might be necessary?

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Cases

• 60yo wm presents with urinary hesitancy. - PSA
• 50yo wf presents with a colon mass. - CEA
• 45yo bf presents with bloating and swollen belly.
  CA125
• 20yo wm presents with gynecomastia. b-HCG and
  AFP

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Cases

• What are you going to check? - biomarkers
• What are you going to follow after resection of
  the malignancy that each has? - biomarkers
• Does anything you check tell you the prognosis of
  their disease? - biomarkers
• Does anything you check affect how aggressively
  you treat them? - biomarkers
• Are you going to follow anything to tell you when
  further treatment might be necessary? - biomarkers

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Biomarkers

• What can you name?
• How did we find them?
• How long have they been around?
• Are there any new ones?

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Beta HCG granddaddy of biomarkers

• First used for pregancy but identified as a tumor
  marker in the 1930s
• Normally secreted by the syncytiotrophoblast to
  help maintain pregnancy by modulating other
  hormone release.
• Associated with trophoblastic or germ cell
  tumors, choriocarcinoma and testicular cancer.

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Alpha-fetoprotein AFP

• As the name suggests is a fetal serum protein.
• Discovered by Bergstrand CG, Czar B.
  Demonstration of a new protein fraction in serum
  from the human fetus. Scand J Clin Lab Invest
  1956 8174.
• Associated with non-seminomatous testicular
  cancer and primary hepatocellular carcinoma but
  also pregnancy, hepatitis and cirrhosis.

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CEA

• 200 kD glycoprotein identified in 1964
• Found normally in embryonic and fetal gut
• Can be elevated in colorectal, breast, lung,
  prostatic, pancreatic, and ovarian carcinoma.
• Primarily used in colorectal cancer

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CEA
Originally identified in 1964 by Ouchterlony gel
diffusion immunoelectrophoresis assays of
immunized and immunotolerized rabbits.
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PSA

• Neutral protease normally produced in the acinar
  cells and ductal epithelium of the prostate.
• Discovered in 1979.
• Associated with any abnormalities in the prostate
  but more consistently elevated in prostate cancer.

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PSA
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CA-125

• High molecular weight glycoprotein normally
  expressed by coelomic epithelium during
  embryogenesis.
• Discovered in 1981.
• Used to follow epithelial ovarian carcinoma
• Named because it was isolated from hybridoma
  OC125 and is a Carbohydrate Antigen (CA)

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CA-125
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CA19-9

• Carbohydrate antigen CA 19-9 is a glycoprotein
  with a sialylated lacto-N-fucopentaose II
  glycosylation. Only expressed by people with
  lewis a or b expression.
• Named from the 1116-NS-19-9 hybridoma clone.
• Associated with a variety of GI malignancies
  especially pancreatic cancer.
• Discovered in 1981.

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CA 19-9
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CA 15-3

• High molecular weight glycoprotein/mucin
  identified in 1984.
• Normally found on the apical side of mammary
  ducts and alveoli.
• Associated with breast cancer.

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CA 15-3
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New age of biomarkers

• Genomics
• Gene expression profiling
• Circulating tumor cells
• Proteomics

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Gene expression profiling
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Circulating Tumor Cells
                                                
                                                  
                                                  
                                                  
      For in vitro diagnostic use. CellSearch
is a trademark of Veridex, LLC. CellSave and
CellTracks are registered trademarks
of Immunicon Corporation, Huntingdon Valley, PA.
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What we are going to do

• Peripheral Blood Molecular Staging of Breast
  Cancer A prospective cohort study designed to
  determine the clinical significance of molecular
  detection of breast cancer in the peripheral
  blood of stage IV breast cancer patients.

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Study participants

• Patients gt21yo with measurable metastatic breast
  cancer starting on a new systemic therapy.
• Must be ECOG 0-2, available and otherwise cancer
  free
• OR
• Volunteer gt21yo with no evidence of malignancy

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Study procedure

• Phlebotomy at enrollment for all participants and
  at weeks 5, 10-12, 16 and at long term follow-up
  for cancer patients.
• Imaging at enrollment, 10 weeks and at long term
  follow-up with CT, MRI or PET-CT for cancer
  patients.
• Appropriate biopsies if any will be obtained at
  baseline and 10 weeks and at physician discretion
  in longer follow-up.

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What are we going to do with the samples from the
120 patients?

• Tumor samples will be both frozen and formalin
  fixed and laser capture microdissection will be
  used to isolate malignant cells.
• Microarrays will be evaluated for changes
  associated with disease resistance and
  susceptibility.

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Imaging

• Will be reviewed by an independent radiologist in
  a randomized and blinded fashion.

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Blood samples

• 7.5 ml Cell Search assay by Veridex for CTC
• 20ml Spun in a porous barrier density gradient
  to enrich for epithelial cells.

Ficoll
Porous barrier density gradient centrifugation
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• Enriched cells will be pelleted and lysed and RNA
  will be isolated for RT-PCR.
• Mam, pip, TFF1, mamB, CK19, muc1, PSE and EpCAM
  RNA levels will be quantitated relative to
  ß2-microglobulin in healthy and cancer patients.
• Results will be assessed for correlation to
  patient response and survival and compared with
  the veridex assay to establish sensitivity of the
  method.

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• Proteomics
• 10ml plasma collected and aliquoted
• 10ml serum collected and aliquoted
• Aliquots of serum from patients with similar
  pathologic type and ER/PR status will be assessed
  by fluorometric labelling with 2D gel
  electrophoresis.
• For responders pretreatment samples will be
  labeled with cy2(blue) and postresponse samples
  with cy5(red) and healthy control sera with
  cy3(green). Spots with above normal expression
  that decrease with treatment will be identified.
• Similarly for progressors Same labeling but
  spots that increase despite treatment will be
  identified.

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Samples 28101(pre-treatment) vs
31491(post-treatment)
3D image for spot number 1341
Cy2 (blue) 28101 Cy5 (red) 31491
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• Spots with differences can be excised and
  identified by mass spectroscopy.
• Method allows 1 ng detection limit.
• Once identified, serum levels can be verified by
  western blot for those proteins with available
  antibodies.
• Suitable proteins identified will then be
  assessed in future studies prospectively and in
  blinded fashions.
• Proteins identified should give some insight into
  the pathophysiology /or treatment of the
  diseases in breast cancer.

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• Machinery should be in place Nov. 1.
• Samples will be sent to Barbara Zehnbauer for the
  CTC samples and to Mark Watson for the genomic
  and proteomics samples
• Study registration is coordinated by Cynthia
  Camillo _at_ 454-8901
• OR
• Angela Cato _at_ 747-9202