Tumor Biomarkers

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Tumor Biomarkers

• Kentucky Cancer Registry
• Fall Conference
• Joe Pulliam, M.D.

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Tumor Markers

• Defined by NCI as substances found in abnormal
  amounts in patients with cancer
• Blood
• Other body fluids
• Other tissues
• Conceptually also applies to physical exam
  findings and radiologic markers (clinical tumor
  markers)

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Tumor Markers

• Some markers are unique for a cancer type
• Others are found in many types of cancers
• Others may also be found in patients without
  cancer but that have other diseases
• Ex. Prostate specific antigen

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Tumor Markers

• Used for
• Diagnosis
• Monitor response to treatment
• Prognosis
• Predict response to particular treatments
• Generally cannot be used alone to diagnose cancer
  must be used with other methods such as biopsy

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How are Tumor Markers selected?

• Basic Research
• To show a potential marker exists
• Evidence-based Translational Research
• To show the markers add value to healthcare
• Practice Guidelines Organizations
• Review the literature and provide standards
• American Society of Clinical Oncology
• National Comprehensive Cancer Network
• National Academy of Clinical Biochemistry
• College of American Pathologists

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How are Tumor Markers selected?
Basic Research
Translational Research and Evidence Based Medicine
Observed Changes in Treatment Outcome
Clinical Practice Guidelines
Changes in Patient Care
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How are Tumor Markers selected?

• Coordinated efforts of cancer staging and
  database centers
• American Joint Commission on Cancer
• International Union for Cancer Control
• Prognostic Factors Task Force
• NCI Surveillance Epidemiology and End Results
• Non-anatomic tumor markers were first added to
  AJCC Cancer Staging Manual, 6th ed. and expanded
  in the 7th ed.

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New Tumor Markers

• Many of the new non-anatomic tumor markers refer
  to components of tumor signaling pathways
• Prognostic significance
• Potential therapeutic targets (predictive markers)

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Ligand
Receptor
Intracellular Signaling Molecules
Cellular Changes
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www.biocarta.com/pathfiles/egfPathway.asp
accessed 9-8-10
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New Tumor BiomarkersHer2 in Breast Cancer

• Human Epidermal Growth Factor Receptor 2
• Nomenclature HUGO designation ERBB2
• aka NEU, NGL, HER2, TKR1, HER-2, c-erb B2,
  Her2/neu
• Cell membrane tyrosine kinase receptor
• Signals after forming homo- or heterodimers with
  other EGFR family members
• Amplified in 18 to 20 of breast cancers

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New Tumor BiomarkersHer2

• Clinical relevance
• Correlates with poor prognosis
• Correlates with resistance to endocrine therapy
• Her2 positive tumors often have lost expression
  of ER/PR
• Predicts response to anthracyclines through
  linkage to TOPO2 gene, with which it is often
  co-amplified
• Predicts response to targeted therapy
  (Trastuzumab/Herceptin)

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New Tumor BiomarkersHer2

• Detection of increased Her2 can be performed in
  several ways
• Immunohistochemistry (IHC)
• Fluorescence in situ hybridization (FISH)
• Chromogenic in situ hybridization (CISH)
• Silver-enhanced in situ hybridization (SISH)
• Quantitative reverse-transcriptase polymerase
  chain reaction (Q RT-PCR)

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Her2 Detection

• Can use DNA, RNA or protein

www.herceptin.com/.../interpreted-results.jsp
accessed 9-8-10
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Her2 by IHC
www.herceptin.com/.../interpreted-results.jsp
accessed 9-8-10
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Her2 by FISH

• This diagram gives the ratio of Her2 signals to a
  chromosome 17 centromeric probe
• FISH can also be done with a single Her2 probe
  and reported as the number of probe signals
  visualized

www.herceptin.com/.../interpreted-results.jsp
accessed 9-8-10
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Her2 by IHC

• Advantages
• Quick, cheap, easy
• Storage for years
• Tumor morphology visible
• FDA approved

• Disadvantages
• Susceptible to variations in testing protocol
• Semiquantitative and subjective score
  interpretation

AJCP 2009 132539
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Her2 by FISH

• Advantages
• Less susceptible to variations in testing
• Score interpretation more objective, quantiative
• Identifies Her2 amplified cases within IHC 2
  cases
• FDA approved

• Disadvantages
• Expensive equipment and probes
• Signal decays with time
• Difficult to see tumor morphology (dark field)

AJCP 2009 132539
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Testing Algorithm for Her2 IHC/FISH
Arch Pathol Lab Med 2007, 13118
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Her2 by CISH or SISH
AJCP 2009 132539
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Her2 by CISH or SISH

• Advantages
• Bright field conventional microscopy
• Stable signal over time
• Can visualize tumor morphology
• DNA more stable analyte than protein

• Disadvantages
• New technique, therefore little experience
• Some discordance with FISH for low level
  amplification

AJCP 2009 132539
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Scarff-Bloom-Richardson (SBR) Grade in Breast
Cancer

• Histologic grading system for breast cancer
• Correlates well with prognosis
• Adopted by World Health Organization in 1968
• Also referred to as Nottingham modification, or
  Bloom-Richardson grading

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Scarff-Bloom-Richardson (SBR) Grade

• Tubule Formation
• Majority of tumor (gt75) 1 point
• Moderate degree (10-75) 2 points
• Little or none (lt10) 3 points
• Nuclear pleomorphism (compare to adjacent normal
  epithelium)
• Small, regular uniform cells 1 point
• Moderately increased size and variability 2
  points
• Marked variation 3 points
• Mitotic Count (must adjust for microscope field)
• Low 1 point
• Moderate 2 points
• High 3 points
• Overall Grade is Sum of Scores
• Grade 1 well differentiated 3 to 5 points
• Grade 2 moderately differentiated 6 to 7
  points
• Grade 3 poorly differentiated 8 to 9 points

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Scarff-Bloom-Richardson (SBR) Grade
Grade 2
Grade 3
Grade 1
Tubule Formation
Nuclear Pleomorphism
Mitotic Count
Adapted from www.breastpathology.info/Special20Ty
pes.html 9-8-10
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Proliferation as a Tumor Marker

• The definition of significantly proliferative
  varies with the type of cancer
• In some tumors, proliferation may be used as a
  marker for diagnosis
• Burkitt non-Hodgkin lymphoma
• In other tumors, proliferation may be correlated
  with prognosis

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Proliferation as a Tumor Marker

• How to measure proliferation
• Mitotic count simplest
• Ex. SBR grade for Breast Cancer
• Mitosis-Karyorrhexis Index
• Neuroblastoma
• Recognizes difficulty distinguishing mitotic
  cells from apoptotic cells
• Immunohistochemistry
• Special proteins expressed during cell cycle
  (usually with DNA synthesis)
• PCNA Proliferating Cell Nuclear Antigen
• Mib1
• Ki-67
• Flow cytometry or Image analysis
• The amount of DNA per cell correlates with phase
  of cell cycle
• Formerly used in breast cancer

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Mib-1 in Burkitt Lymphoma
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Multigene Signature Methods

• Most commonly used in Breast cancer
• Will likely become important in other tumors
• Gene expression microarray
• Include several pathways of significance for each
  type of cancer
• Correlate with distinct biological subtypes
• Methods
• Mammaprint 70 gene
• Oncotype Dx 21 gene
• others

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Multigene Signature Methods

• Derived from basic research and translational
  research using tumor biospecimens correlated with
  tumor registry outcome data
• Potential tumor markers validated in subsequent
  testing

NEJM 2009, 360790
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Multigene Signature Methods

• 4 molecular breast cancer subtypes
• Basal-like
• Luminal A
• Luminal B
• Her2-like
• Good correlation with standard pathological
  variables
• Potential insight into treatment failures or new
  therapeutic interventions

NEJM 2009, 360790
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Mammaprint

• Gene signature derived from selected
  retrospective review
• 78 node negative breast cancer patients not
  treated with adjuvant therapy
• Supervised top-down approach
• Two outcomes Low Risk or High Risk of
  disease recurrence without adjuvant therapy
• Uses fresh or frozen tumor, not formalin-fixed
  paraffin-embedded
• 70 gene cDNA microarray
• FDA approved

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www.nature.com/.../v17/n7/fig_tab/2402974f1.html
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Oncotype Dx

• Uses a candidate-gene approach to measure gene
  expression by Q-RT-PCR

http//www.oncotypedx.com/en/Breast/HealthcareProf
essional/Underlying.aspx accessed 9-8-10
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Oncotype Dx

• Uses formalin-fixed, paraffin-embedded tissue
• Normalized gene ratios by comparing 16 cancer
  genes to 5 reference genes
• this also corrects for variable RNA quality
  between samples

Nature Protocols 1, 1559 - 1582 (2006)
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Oncotype Dx

• To be used in estrogen/progesterone receptor
  positive patients
• Informative for node positive patients
• Not FDA approved
• Endorsed by ASCO and NCCN Clinical Practice
  Guidelines
• Recurrence Score scale from 0 to 100
• Could be thought of as will patient benefit from
  traditional chemotherapy score
• Low 0-18 low chance of recurrence and no
  benefit from traditional chemotherapy
• High 31 or more high chance of recurrence and
  favorable response to traditional chemotherapy

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Multigene Signature Methods

• Both Mammaprint and Oncotype Dx are undergoing
  prospective clinical trials
• Mammaprint MINDACT
• will also assess utility in patients with up to 3
  positive lymph nodes
• Oncotype Dx TAILORx
• designed to further assess utility of
  intermediate risk scores for treatment decisions

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New Tumor BiomarkersKRAS in Colorectal Cancer

• Intracellular second messenger signaling molecule
• Relays messages from receptor tyrosine kinases
• Other family members include NRAS and HRAS
• One of the most frequently mutated oncogenes in a
  variety of cancers
• Activating mutations in 35-45 of colorectal
  carcinoma
• Confer resistance to EGFR-inhibitor drugs

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Progression free survival
Overall survival
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Spectrum of RAS mutation in Colorectal Cancer

• Most are activating mutations in codons 12 and 13
  (35 of CRC cases)
• Initial trials showing resistance to
  EGFR-inhibitors looked at only these codons
• Other mutations (5-10 of CRC cases) are also
  activating and confer resistance to
  EGFR-inhibitors
• Codons 14, 17, 61, 146
• Activating NRAS mutations (3 of CRC cases also
  confer resistance

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EGFR-RAS-PI3K Signaling Pathways in Colorectal
Cancer
World J Gastroenterol 2010, 161177 accessed
9-8-10
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Future Tumor Markers in Colorectal Cancer?

• KRAS and BRAF now
• PIK3CA, and PTEN in future (Quadruple Negative
  CRC)?
• Also NRAS (Lancet Oncol, 2010 Aug, 11753)

J Clin Oncol 2010, 281254 accessed 6-4-10
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New Tumor BiomarkersKIT in Gastrointestinal
Stromal Sarcoma (GIST)

• GIST uncommon tumor thought to arise from
  interstitial cells of Cajal (cells of the
  autonomic nervous system)
• Most GIST tumors have an activating in a receptor
  tyrosine kinase
• KIT mutation in 50-85 of GIST
• KIT also known as CD117 or stem cell factor
  receptor
• Nearly all KIT mutations in GIST are in exon 11,
  some in exon 9 or 13, rarely in exon 17
• PDGFR-alpha mutation in 10-20 of GIST

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New Tumor BiomarkersKIT in Gastrointestinal
Stromal Sarcoma (GIST)

• Fortunately, nearly all KIT and PDGFR-alpha
  mutations in GIST respond to treatment with
  Gleevec (a tyrosine kinase inhibitor)
• Rare exon 17 mutations
• Detection by IHC sufficient
• Activating KIT mutations also present in
  melanoma, mastocytosis, acute myeloid leukemia,
  myeloproliferative neoplasms
• However, these frequently have exon 17 mutations
  resistant to Gleevec, so mutation analysis
  required

PET images before and after 8 weeks of Imatinib
for GIST
Oncologist, 2006, 119
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Activating KIT mutations
http//atlasgeneticsoncology.org//Genes/KITID127.h
tml accessed 9-8-10
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KIT Immunohistochemistry
commons.wikimedia.org/wiki/FileGastric_GIST_...
accessed 9-8-10
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New Tumor BiomarkersJAK2 in Myeloproliferative
Neoplasms

• Myeloproliferative Neoplasms
• Class of hematologic cancers in which there is a
  neoplastic proliferation of maturing blood and
  marrow cells
• Frequently associated with marrow fibrosis
• Often characterized by specific mutations
• Knowledge of these mutations led World Health
  Organization to revise its diagnostic criteria
  for myeloproliferative neoplasms
• Hopefully this knowledge will lead to targeted
  therapies

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New Tumor BiomarkersJAK2 in Myeloproliferative
Neoplasms

• Diagnostic mutations in myeloproliferative
  neoplasms
• JAK2
• Polycythemia Vera erythroid cells
• 90 of P. vera have JAK2 V617F mutations, with
  other JAK2 mutations identified by sequencing
• JAK2 or MPL
• Essential Thrombocythemia platelets and
  megakaryocytes
• Primary Myelofibrosis
• Both have JAK2 V617F mutation in 50-60 of cases,
  MPL W151L/K mutations in 10-15
• KIT
• Mastocytosis mast cells
• BCR-ABL
• Chronic myelogenous leukemia myeloid cells
• PDGFR-alpha, PDGFR-beta, FGFR1
• Myeloid neoplasms with eosinophilia

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MPL-JAK2 Signaling Pathway

• JAK2 is a second messanger for a variety of
  growth factor receptors
• Erythropoietin receptor
• Thrombopoietin receptor (MPL)
• Various other cytokine receptors

Nature Reviews Cancer 7, 673-683 (September 2007)
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Summary

• Tumor markers
• Arise from basic and translational research,
  clinical practice guidelines, which build on data
  from tumor registries
• Those shown to be significant for management have
  been added to the Site Specific Factors list in
  the SEER required database
• Many new tumor markers are within tumor signaling
  pathways and are amenable to therapeutic
  intervention
• We can expect more tumor markers to be added in
  the future

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From Reita Pardee

• Her2 IHC
• Her2 FISH
• Her2 CISH
• Oncotype
• Mammaprint
• Markers of proliferation (mitotic count)
• KRAS
• KIT (IHC)
• JAK-2
• CEA
• CA19-9
• Alpha Fetoprotein