1. Research Presentation
Jason M. Leibowitz, MD
June 25, 2009
Preceptor: Marcia S. Brose, MD PhD
Otorhinolaryngology: Head and Neck Surgery at PENN
Excellence in Patient Care, Education and Research since 1870

2. Overview Background Hypothesis Methods Results Discussion
Conclusions & Future Directions

3. Thyroid Cancer in the United States
Thyroid cancer is the most common endocrine neoplasm.
Thyroid cancer will be diagnosed in 33,550 individuals (8070 men and 25,480 women) this year.
From incidence of thyroid cancer increased by 6.2% mostly due to increased detection.
From 1985 to 2004 mortality rate increased by 0.3% a year.

4. RAI-Refractory Disease
25-50% of metastatic thyroid cancers lose ability to take up Iodine.
Iodine Uptake inversely correlates with survival.
This is attributed to down regulation of the Na+/I- Symporter (NIS).
Limited treatment options for unresectable thyroid cancer refractory to RAI.
Loss of Iodine uptake correlates inversely with survival
This is mostly due to increasing methylation of the NIS promoter.
Once TSH, RAI is ineffective, and XRT has already been maxed, or pt has distant disease, what do we have to offer….?

5. Molecular Changes in Thyroid Cancer
Much of the recent progress owes itself to the finding that a large degree of thyroid cancers harbor genetic alterations.
Many of the genetic abnormalities lead to altered cell signaling.

6. Molecular Pathway involved in Thyroid Cancer
Activation of MAPK pathway
Oncogenic activation of this pathway in 70% of all thyroid cancers.
BRAF is a serine threonine kinase
BRAF is an intracellular effectors of MAPK pathway
Activated by RAS binding & recruitment to the cell membrane
Phosphorylates and activates MEK --> activates ERK
This signaling pathway is pivotal in he development of both PTC & FTC.
Schematic illustration of the MAPK pathway. The signaling starts at the cell membrane receptor upon stimulation by extracellular mitogenic signals (e.g., growth factors). Upon activation by binding with GTP, the Ras protein interacts with and activates Raf protein kinase. The B-type Raf kinase, or BRAF, is the most abundant and potent in the Raf family in many cells and is shown here in the figure. Activated BRAF phosphorylates and activates two MEKs, MEK1 and MEK2. Activated MEK1/2 in turn phosphorylates and activates the two immediately downstream ERKs, ERK1 and ERK2. ERK1/2 subsequently phosphorylates downstream proteins, many of which are kinases themselves, ultimately leading to alterations in the expression of various genes in the nucleus involved in cell proliferation, growth, survival, and tumorigenesis. Unique to some PTC is also the occurrence of RET/PTC, a recombinant protein consisting of the tyrosine kinase domain of the RET, a membrane receptor tyrosine kinase, and a portion of an unrelated protein. RET/PTC can activate the MAPK pathway through a step upstream of Ras.
Xing, 2007.

7. BRAF V600E in Thyroid Cancer
2003: The BRAF V600E mutation is the most common genetic alteration in thyroid cancer, occurring in about 45% of sporadic papillary thyroid cancers (PTCs).
2002 BRAF mutations were discovered in Melanoma
in 2003 found in WDTC, primarily PTC.
Nucleotide > Valine to Glutamate substitution (activation loop--> constitutive activation of BRAF kinase and hyperphophorylation of MEK & ERK)
V600E leads to constitutive activation of BRAF kinase
More common in classic and tall cell-variant, less common in follicular-variant
24% of PTC-derived ATC

8. BRAF V600E Point mutation in 40-45% of PTC
Upregulation of MMP, VEGF --> invasion, angiogenesis
Silencing of tumor suppressive genes, genes involved in iodine transport
BRAF mutation associated with multiple negative prognostic indicators.
V600E = valine to gutamate substitution
BRAF mutation also found in 15-20% of poorly diff/anaplastic thyroid cancer.
Picture is B-RAF protein structure

9. RAS
Family of small G-proteins involved in transduction of cellular signals from the cell membrane.
Mutations in RAS gene lead to inappropriate activation with constitutively activated downstream pathways and also promote chromosomal instability.
20% FTC contain a RAS mutation RAS mutations may correlate with aggressive behavior (tumor dedifferentiation and poorer prognosis).
Ras gene encodes a G-protein located at the inner surface of the cell membrane.
Transduce signals from cell membrane tyrosine kinases and G- protein coupled receptors
Activates MAPK and other signalling pathways--- PI3K/AKT
Active RAS usually quickly becomes inactivated by its intrinsic GTPase activity and due to cytoplasmic GTPase activity--> point mutations in RAS lead to increased GTP affinity or inactivation of auto-GTP-ase
RAS mutations also occur on benign follicualr adenoma
RAS mutation: 10% PTC, 35% poorly diff, 55% anaplastic
PTC with follicular features almost always have the RAS mutation

10. Targeted Therapy in Thyroid cancer
Loss of differentiation (inability to trap RAI), unresectable lesion, leads to poor prognosis
BRAF inhibitors
BAY (Sorafenib)
Multikinase inhibitor

11. Sorafenib Orally active multikinase inhibitor (study dose 400mg BID).
Monoclonal antibody with multiple targets including BRAF, VEGFR1, VEGFR2.
Blocks tumor cell proliferation and angiogenesis.
FDA approved for treatment of RCC and hepatocellular carcinoma.

12. Targeted Therapy and Genotype
K-RAS gene mutation and metastatic colorectal carcinoma.
Recent results from Phase II & III clinical trials demonstrate that patients with metastatic colorectal cancer benefit from anti-EGFR therapy.
Patients with K-RAS mutation in codon 12 & 13 should not receive anti-EGFR therapy since they do not receive any benefit.
EGFR and non-small cell lung cancer:
Epithelial growth factor receptor
10% mutated in NSCLC
EGFR mutations are predictors of TKIs responsiveness and may show a long lasting response to TKIs
EXON 19 Deletion respond better to TKIs.
ASCO currently recommends testing to predict response to anti-EGFR monoclonal antibody therapy - except if mutation is in codon 12/13
Non-small cell lung cancer, key components of cell signaling are abnormal -- lead to cell proliferation and differentiation --- components of these signaling pathways a key targets for targeted therapies. EGFR is strongly associated with malignant proliferation and adverse prognosis. EGFR is a receptor tyrosine kinase. Mutated --> targeted therapies include monoclonal antibodies against extracellular domain such as cetuximab and matuzumab and TK inhibitors such as gefitinib & erlotinib. Exon 19 deletions respond better to TKI’s.

13. Prior Data 84 weeks N=43 WDTC N= 52
POINT: Sorafenib associated with prolonged stable dz and modest tumor shrinkage in solid tumors.
Kaplan-Meier survival curve for PFS in pt in clinical trial treated with sorafanib -- Everyone-- all 52 subjects -- PFS 84 weeks
Curve 2: PFS Diff Thyroid cancer, 43 pt, PFS not reached
Phase II trial -- JCO Oct > phase 2, N=30, partial response 23.3%, stable disease rate 53.3%; majority were wdtc, 2 pt with poorly/anaplastic had dz progresson
Progression Free Survival for all 52 patients---Mean is 84 weeks (50% 84 weeks)
NO DEATHES
Statician used -- appropriately powered
PFS = The length of time during and after treatment in which a patient is living with a disease that does not get worse. Progression-free survival may be used in a clinical study or trial to help find out how well a new treatment works.
84 weeks
N=43
WDTC
N= 52

14. Papillary vs. Follicular
43 patients FTC, 24 PTC
Statistically not significant although there was a trend -- probably due to immature data-- it is early data, and if we follow it out, it will be statistically significant (chi squared p<0.095
FTC is not equal to PTC
FTC PFS not reached,
PTC 72 weeks
FTC = 19
PTC= 24
P<0.095

15. Prior Data Conclusions from prior data: Improved PFS with Sorafenib.
Improved PFS of FTC treated with Sorafenib when compared to PTC.

16. Overview Background Hypothesis Methods Results Discussion
Conclusions & Future Directions

17. Hypothesis
There are specific genotypes (i.e. BRAF V600E, RAS mutations) that predict favorable response to targeted therapy (Sorafenib).

18. Null Hypothesis
Specific genetic mutations do not predict response to targeted therapy in thyroid cancer.
I.E prior cohort is random and all pts respond the same to Sorafenib

19. Overview Background Hypothesis Methods Results Discussion
Conclusions & Future Directions

20. Research Plan
Tissue samples collected from patients with treatment-resistant thyroid cancer with long term follow-up (approximately 30 patients).
All patients received targeted therapy (Sorafenib).
Samples with WDTC analyzed for mutations in BRAF and RAS genes when available:
BRAF - V600E
RAS - Exon 12, 13, 61

21. RESULTS

22. Sequence Output
Computer program interprets data and produces an electropherogram, (aka trace)
Each peak represents a base:
A = Adenosine
T = Thymine
C = Cytosine
G = Guanine
N = Reading cannot be determined

23. Overview Background Hypothesis Methods Results Discussion
Conclusions & Future Directions

24. Results of Stage 1 Analysis
M = F = 15
PTC=17, FTC= 9, Other (ATC/PD, MTC): 4
Samples analyzed for BRAF mutation:
23/30 (76.6%): samples analyzed for BRAF mutation
4/30 (13%): definite genotype but questioned due to phenotype (ATC/PD, MTC)
2/30 (6%): unable to amplify DNA despite multiple PCR attempts
1/30 (3%): pending analysis
18/30 samples analyzed for RAS mutation, all WT copies of the gene
First 30 patients with long term data evaluated
Male=female, but vast majority are DTC
Majority were differentiated thyroid cancer FTC and PTC
4. Few had adriamycin prior to study
WHAT’s important is that all of the RAS genes were wild-type --> so it does not seem that an underlying mutation in RAS is the underlying molecular mechanism for the improved PFS seen in FTC-- so what is this mechanism

25. Results of Stage 1 Analysis
N=22 (interim analysis)
13 WT BRAF
9 BRAF V600E
16 PTC
9 WT BRAF, 7 V600E
6 FTC
4 WT BRAF, 2 V600E

26. BRAF V600E P<0.02 N=13 (WT=8, V600E=5)
13 patients --> 8WT, 5 BRAF V600E
Statistically significant chi-squared p < 0.02
WT PFS 54 weeks
V600E PFS not reached

27. Updated genetics
In our expanded analysis to 22 pts with WDTC, the effect is no longer significant but the trend exists.
We are further investigating BRAF copy number in these patients
N =22
WT = 13
BRAF V600E = 9
p=NS
We reported data on 13 patients that showed improved PFS in pts with BRAF V600E
In our expanded analysis to 22 pts with DTC, the effect is no longer significant but the trend exists.
We are further investigating BRAF copy number in these patients- we think that this is reason that we no longer see the effect _ (I.e. some of WT BRAF patients are actually overexpressing the BRAF protein and are thus like the mutants.-- more on this later- WHAT’s important is that all of the RAS genes were wild-type --> so it does not seem that an underlying mutation in RAS is the underlying molecular mechanism for the improved PFS seen in FTC-- so what is this mechanism

28. Overview Background Hypothesis Methods Results Discussion
Conclusions & Future Directions

29. BRAFV600E Correlates with worse Survival
Elisei et. al, J Clin Endocrinol Metab, October 2008, 93(10):3943–3949

30. BRAFV600E Correlates with worse Survival State of the mutation in PTC, 10/2008
However Our BRAF data is important given in a retrospective analysis of patients from the pre-kinase era, BRAFV600E patients have done worse.
Traditionally BAF V600E correlated with a worse survival and poorer outcome in WDTC
This directly contradicts our recent findings

31. THE BRAF connection Ciampi et al. 2005
As we showed before, upon further analysis the difference between BRAF WT & V600E has lost statistical significance as we have increased our number of specimens. It is possible that this is due to chance, and that with the completion of all 50+ specimens the statistical significance will return. However another possibility is copy number gain, especially in FTC, may account for this difference. This was first shown in 2005 by Ciampi et al. They looked at 62 FTC, 32 PTC --> they found that although rare in PTC, copy number gain is quite common in FTC/ Hurthle cell neoplasms. (16-45%) Copy number gain involves either amplification of the BRAF gene or increased number of copies of chromosome 7. Shown above is a slide if FISH (fluorescence in situ hybridization) with a BRAF specific and Chromosome 7 specific probe. There data has shown that FTC with copy number gain is more widely invasive, thus demonstrating a worse prognosis. This occurs because higher amounts of the BRAF gene product are expressed, thus mimicking the V600E mutation. They did not see an overlap in copy number gain with RAS mutations. This may explain the fact that we did not detect any RAS mutations in our samples.
THE KEY is COPY NUMBER GAIN OF BRAF (IN FTC) - would have normal WT BRAF gene but extra copies
WOULD HAVE A WT BRAF IN THESE CASES
ALSO, THEY DID NOT SEE AN OVERLAP IN RAS MUTATION AND BRAF COPY NUMBER
Ciampi et al. 2005

32. Updated genetics
In our expanded analysis to 22 pts with WDTC, the effect is no longer significant but the trend exists.
We are further investigating BRAF copy number in these patients
N =22
WT = 13
BRAF V600E = 9
p=NS
We reported data on 13 patients that showed improved PFS in pts with BRAF V600E
In our expanded analysis to 22 pts with DTC, the effect is no longer significant but the trend exists.
We are further investigating BRAF copy number in these patients- we think that this is reason that we no longer see the effect _ (I.e. some of WT BRAF patients are actually overexpressing the BRAF protein and are thus like the mutants.-- more on this later- WHAT’s important is that all of the RAS genes were wild-type --> so it does not seem that an underlying mutation in RAS is the underlying molecular mechanism for the improved PFS seen in FTC-- so what is this mechanism

33. BRAF (red) x 3
7 centromere (green) x 3

34. BRAF x4
7 centromere x4

35. 4 copies each
3 copies each

36. THE BRAF connection! Positive Predictor!
Ciampi et al, 2005.

37. Summary
Good progression free survival in patients treated with Sorafenib.
BRAF V600E appears to predict for improved outcome in patients treated with sorafenib.
BRAF copy number gain may explain improved outcome of patients with FTC over patients with PTC

38. Future Directions Completion of genotyping analysis of all patients
Evaluation of copy number gains in WDTC
Hypothesis: Copy number gain accounts for improved survival in FTC treated with Sorafenib
Null: Copy number gain does not influence survival in FTC

39. Selected Sources
Ciampi R, Zhu Z, Nikiforov YE. BRAF copy number gain in thyroid tumors detected by fluorescence in situ hybridization. Endocrine Pathology 2005; 16(2):
Ciampi R, Nikiforov YE. Alterations of the BRAF gene in thyroid tumors. Endocrine Pathology 2005; 16:3):
Gupta-Abramson V, Troxel AB, Nellore A, et al. Phase II Trial of Sorafenib in Advanced Thyroid Cancer. Journal Clin Onc 2008; 26 (29):
Kundra P, Burman KD. Thyroid Cancer Molecular Signaling Pathways and Use of Targeted Therapy. Endoc Metab Clin N Am 2007;36:
Murer B. Targeted Therapy in Non-Small Cell Lung Cancer. Arch Path Lab Med. 2008; 132:
Nikiforov YE. Thyroid Carcinoma: Molecular Pathways and Therapeutic targets. Modern Pathology 2008; 21: S37-S43.
Vasko V, Ferrand M, Cristofaro JD et al. Specific Pattern of RAS Oncogene Mutations in Follicular Thyroid Tumors. J. Clin Endocrin. & Metab. 2003; 88(6):
Xing M. BRAF Mutation in Papillary Thyroid Cancer: Pathogenic Role, Molecular Basis, and Clinical Implication. End Rev 2007; 28(7):

40. Thanks
Marcia Brose, MD PhD
Cathy Ma MD, PhD
Kanchan Puttaswamy, MS