April 9, 2011
Diagnostic Molecular Pathology Update
Christopher D. Gocke, M.D.Associate Professor of Pathology & Oncology Johns Hopkins Medical InstitutionsBaltimore, MD
Disclosures
Christopher D. Gocke, M.D. has no relevant disclosures
Recent advances
• EGFR testing in lung cancer
• New HPV testing recommendations
• Expression analysis of breast cancer
• Warfarin side effects prediction
• HCV genotyping
• Molecular screening for MRSA
• MPDs defined by JAK2 mutation
EGFR TESTING IN LUNG CANCER
The EGFR tyrosine kinase pathwayErbB receptor family:
ERBB1 (EGFR)
ERBB2 (HER2)
ERBB3
ERBB4
Mod Path (2008) 21:S16
These form homo- or heterodimersThese form only heterodimers
EGFR in lung cancer
• NSCLC (80% of all lung cancers) is a clinical, morphologic and genetic mixed bag
• Historically, EGFR is over-expressed in 62% and correlates with poor prognosis
• Ligands are also over-expressed autocrine loop hyperactivity
• Small molecule inhibitors and antibodies
EGFR therapy in lung cancer
gefitinib and erlotinib reversibly block the kinase region of the ERBB proteins
Nature Rev Drug Disc (2004) 3:1001
Erlotinib
Hydrophobic pocket
EGFR inhibitor therapy
• Early (phase II) trials– Response in previously treated patients:
9-19%, median survival 6-8.4 mo (no benefit over chemo alone)
• But, some subgroups did better– Women (19% response vs 3% men)– Adenocarcinoma histology– Asian ethnicity– Never smoker (36% vs 8%)
Why the better response?
• Acquired, somatic mutations in EGFR– Most responding patients had
heterozygous mutations– Mutations result in increased receptor
activation (phosphorylation)
NEJM (2004) 350:2129
Mutations are clinically significant
• Gain-of-function mutations in 77% of responders vs 7% of refractory patients
• 10-20% responders without EGFR mutations indicate other molecular causes (e.g., EGFR amplification or mutations in other ERBB family members
EGFR status(N= 487)
Response rates (%)
PFS (mo) OS (mo)
WT 5 – 14 1.7 – 3.6 4.9 – 8.4
Mutant (34%) 16 – 84 9.9 – 21.7 13 – 30.5
EGFR mutations
Nat Rev Cancer (2007)7:169
Resistance to EGFR inhibitors
• Develops after 6-12 months in most
• About ½ acquire a T790 exon 20 mutation
• Altered EGFR trafficking, active drug excretion are possible alternatives
• 2nd generation drugs are targeting irreversible EGFR binding and other ERBB family members
Current recommendations for molecular assays
• FFPE tissue of adenocarcinoma only (but not mucinous BAC); may need microdissection
• Direct sequencing is gold standard, but mutation-specific methods may be OK
• Several replicates should be run• Heterogeneity exists in tumor and
metastasis—be aware
JCO (2008)26:983
Direct sequencing of EGFR mutations
NEJM (2004)350:2129
Other considerations for EGFR testing
• EGFR mutant pts treated with placebo do better than controls—a prognostic marker
• IHC for protein positive in 50-90% of NSCLC—1 study (BR.21) suggests survival benefit—very inconsistent scoring
• Amplification by FISH/RT-PCR positive in 31-45%—significant overlap with mutation positive—unclear how important
• KRAS and EGFR mutations mutually exclusive—KRAS positive are inhibitor resistant
NEW HPV RECOMMENDATIONS
Infectious disease testing: Improving the Pap smear
• Relative risk of developing high grade dysplasia (premalignancy) if infected with high risk HPV is increased 76-fold
Univ Utah
Guidelines for management of abnormal cervical screening tests
• Consensus statement guided by American Society for Colposcopy and Cervical Pathology
• Am J Obstet Gynecol (2007) 197:346
• Only testing for high-risk (oncogenic) types of HPV is indicated
Infectious disease testing: Improving the Pap smear
• Hybrid capture HPV detection (Digene)
1. Lyse cervical cells 2. Hybridize with cRNA
3. Bind with anti-DNA/RNA
antibodies
4. Add labeled anti-DNA/RNA antibodies
5. Detect label
Management of women withASC-US
• Reflex testing (of the ASC-US specimen) for HPV (preferred on cost basis) OR repeat cytology OR colpo
• Except: adolescents (≤20 yo), no HPV
Management of women with ASC-H
• Colposcopy– If negative (no CIN2,3 or greater):
• HPV testing at 12 mo OR• Cytology at 6 and 12 mo
• Repeat colposcopy if these don’t normalize
Management of women with LSIL
• LSIL is a good indicator of high-risk HPV infection (76.6%)– Prevalence of CIN2+ in this group is 12-16%
• Colpo with biopsy– If negative, manage as for ASC (HPV testing at
12 mo OR cytology at 6 and 12 mo)
• Except:– Adolescents: no HPV testing– Postmenopausal: monitor with HPV test or
cytology, or go direct to colpo
Management of women withHSIL
• High rate of HPV positivity and CIN2 or greater (84-97% by LEEP)
• Colpo with biopsy—HPV testing plays no role in these patients
Management of women withatypical glandular cells (AGC)
• High rate of CIN2+ and invasive cancer (3-17%)
• HPV testing should be done at colpo, but not as a part of triage– If these women are biopsy negative:
• And HPV+, repeat HPV/cytology at 6 mo• And HPV-, repeat HPV/cytology at 12 mo• And HPV unknown, repeat HPV/cyto at 6 mo
Screening
• Most HPV infections clear spontaneously, therefore only women ≥30 yr should undergo HPV testing
Detection of CIN2+ in 35+ y.o. womenPooled test parameters (Int J Cancer (2006)119:1095)
Sensitivity (%) Specificity (%)
HPV testing 94 93
Cytology 60 97
Screening (2)
• Final guidance on 2 controversial areas:– What is the appropriate interval for
rescreening a cyto-/HPV- patient?
3 years (compare to annual Paps); <2% develop CIN3+ over next 10 years (JNCI (2005) 97:1072)
Screening (3)
– What to do with cyto-/HPV+ patients?• A common phenomenon (58% HPV+ women
were cyto- in Kaiser study)• Most (60%) of HPV+ women become HPV-
spontaneously over 6 months (BJC (2001) 89:1616)
• Risk of CIN2+ in them is 2.4-5.1%
Follow-up testing in 12 mo, with repeat positives going to colpo
EXPRESSION ANALYSIS OF BREAST CANCER
Current management guidelines for breast cancer
Not uniform consensus
NCCN BINV-6
• Quick way of examining simultaneous expression of thousands of genes
• Several uses:– Class finding (unsupervised clustering):
• Basal-like (ER/PR/HER2 negative)• Luminal A & B• HER2 over-expressing• Normal-like
(These classes correlate with outcome and response to therapy, but not beyond standard clinicopathologic markers)
Gene expression array
Clinically aggressive
Gene expression array (2)
– Identification of molecular targets for specific therapy (e.g., androgen receptor pathway?)
– Prognostication (supervised analysis)• Is there a pattern that separates 2 different pre-
identified populations, e.g., metastasis-free survival vs not, or cancer recurrence vs not?
Gene expression array (3)
Harvest RNA, label Hybridize Wash
Read intensity Aggregate data
Gene expression array (4)
• Problems– Tissue requirements: prefer frozen tissue– Small sample size for validation (hundreds)– Population bias– Gene list instability
• E.g., 2 different signatures (Mammaprint and Rotterdam) share only 3 genes, yet identify the same groups of low- and high-risk patients
21 gene RT-PCR assay (Oncotype Dx)
• Not an array, but a specific gene panel
• FDA exempt (CLIA) for node negative, ER +, tamoxifen-treated
• Provides a “recurrence score” from 1-100
• Requires FFPE tissue
• Performed at company lab in California
JCO (2008) 26:721
21 gene RT-PCR assay
RS ≥31
RS <18
Prognostic
Predictive
70-gene signature (MammaPrint)
• FDA approved for Stage I-II, node-negative, ≤5 cm invasive tumor, ≤60 yr
• Predicts risk of distant metastasis in next 5-10 yr
• First IVDMIA cleared by FDA (2/07)
• Requires fresh preserved tumor with a minimum % tumor
• Performed at company lab in Amsterdam
MammaPrint signature outcome (without adjuvant therapy)
“Good” = 87% @ 10 yr
“Bad” = 44% @ 10 yr
NEJM (2002) 347:1999
Comparison of commercially available tests
Oncotype DX MammaPrint
Starting material FFPE Fresh mRNA
Number of genes 21 70
Rank of importance of pathways
1) Proliferation2) ER3) HER2
1) Proliferation2) ER3) HER2
Current indication Node -, ER + Node +/-, ER +/-
Eligible patients Older Young and old
Prognostic vs predictive
Prognostic and predictive
Prognostic
Outcome prediction Continuous Dichotomous
Cost effectiveness? In 1 study ?
US FDA status Exempt Approved
Clinical trial TAILORx MINDACT
Study question Who with intermed. risk will respond to chemo?
Who will have good outcome without chemo?
Cost ~$3600 ~$3200
Oncologist (2008) 13:477
WARFARIN MANAGEMENT
Maintenance dose variability
Molec Interv (2006) 6:223
Warfarin metabolism
Molec Interv (2006) 6:223
Genetic variation in warfarin related enzymes
• CYP2C9 (hepatic metabolizer):– *1 (wild-type), rapid metabolizer, ~80%
Caucasian population– *2 and *3, slow metabolizers, 12.2% and
7.9% allele frequencies—heterozygotes take twice or three times as long to achieve steady-state when their daily dose is adjusted down
Genetic variation in warfarin related enzymes
• VKORC1 (Vit K production) (transcriptional variants)– B (wild-type), high-dose phenotype, 60%
allele frequency in Caucasian (wide variation reported)
– A, low-dose, 40% Caucasian (most Asians)
Clinical variability in warfarin dosing
Genet Med (2008) 10:89
FDA action on warfarin
• 2006: adds a “black box” warning for Coumadin about risk of severe or fatal bleeding
• 8/2007: adds notice that genetic variation in CYP2C9 and VKOR1 influence phenotype
• 9/2007: clears Nanosphere’s Verigene test for identification of some alleles
Nanogold DNA genotyping technology
Science (2000) 289:1757
Problems with genotyping for warfarin dosing
• Most studies use INR as endpoint; little data from randomized studies on direct clinical utility in predicting/preventing bleeding or thrombosis (under-anticoagulation)
• Little data in non-Caucasians, children
• How to present dosing advice to clinicians
• Ethical, legal, social implications
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