Molecular testing in indeterminate thyroid nodules

1. MOLECULAR DIAGNOSIS IN
INDETERMINATE THYROID NODULES
- UPDATE 2019 -

2. Fine needle aspiration (FNA) cytology fails to provide a conclusive diagnosis in a subset of
thyroid lesions labeled as “indeterminate” (Thy3a-f, Bethesda III-IV, TIR3a -b)
INDETERMINATE LESIONS
Risk of malignancy is variable among different centers (20%), ranging from 6 to 48%.

3. Italian Classification (SIAPEC 2014)
INDETERMINATE LESIONS

4. INDETERMINATE LESIONS

5. USEFUL TECHNIQUES
Elastography: could be complementary in describing such lesions.
Core needle biopsy: accurate second-line test in thyroid TIR1 (not TIR3).
Immunochemistry: Galectin-3, Cytokeratin-19, and HBME-1.
Trimboli, Endocrine 2015

6. Ultrasonography is a practical and accurate tool in the risk stratification of (such) nodules.
ULTRASOUND
Thyroid ultrasound risk stratification system:
• Thyroid Imaging Reporting and Data System (TIRADS)
• American Thyroid Association (ATA)
• American Association Clinical Endocrinologists/American College of
Endocrinologists and Associazione Medici Endocrinologi (AACE/ACE/AME)
• British Thyroid Association (BTA)
• European Thyroid Association (ETA) (EU-TIRADS)

7. ULTRASOUND
Some studies looked at the accuracy of international risk stratification systems in thyroid
lesions classified as indeterminate.
Ultrasonographic features are a primary filter to select the most appropriate nodule to test.

8. MOLECULAR TESTING

9. MUTATIONS
Thyroid cancer, like all cancers, is a disease of the genome.
Initiation/progression of carcer due to the accumulation of genetic/epigenetic changes:
- somatic mutations
- chromosomal rearrangements
- alterations in gene expression
- microRNA dysregulations

10. MUTATIONS
Genetic alterations occur in the MAP kinase (MAPK) and PI3K/AKT pathways:
- BRAFV600E and RAS point mutations
- RET/PTC and PAX8/PPARγ rearrangement genes.
The Cancer Genome Atlas has reduced the fraction of PTC cases with unknown oncogenic
driver from 25% to 3.5%, offering a high potential for molecular diagnostics.
70% of known genetic
alterations in DTC

11. MOLECULAR TESTING

12. MOLECULAR TESTING
Four commercially available molecular tests to improve risk stratification of thyroid lesions.
Ferraz, Arch Endocrinol Metab 2018
(2012)
(Veracyte, San
Francisco, California)
(2014)
(CBLPath, New York, and
University of Pittsburgh,
Pennsylvania)
(2017)
(Rosetta Genomics, Inc,
Philadelphia, Pennsylvania)
(2015)
(Interpace Diagnostics, Parsippany,
New Jersey)
Plus ThyroPrint (GeneproDX, Santiago, Chile) *.
Mir-THYpe (ONKOS Diagnosticos Molecular LDTA), Ribeirao Preto, Brazil)*.
* Not certified by the College of American Pathologist yet.

13. MOLECULAR TESTING
Ferraz, Arch Endocrinol Metab 2018
Tests commercially available should:
1) Identify and define the mutation panel or the classifier using a training set.
2) Validate the panel/classifier on a validation set.
3) Validate the panel/classifier in prospective, multicenter, independent studies.

14. MOLECULAR TESTING
Ferraz, Arch Endocrinol Metab 2018
(18) (3)

15. MOLECULAR TESTING
Nishino, Cancer Cytopatology 2016
Rule-out Test
Test to predict benign nodules (rule out),
requires high NPV (at least 96% as TIR2)
Rule-in Test
Test to predict malignancy (rule in),
requires high PPV (at least 98 % as TIR5).

16. MOLECULAR TESTING
Ferraz, Arch Endocrinol Metab 2018
Rule out Rule in

17. Effect of prevalence change on test predictive value. Prevalence strongly influences:
- PPV (dashed line): the ability of a test to “rule in” a disease correctly.
- NPV (solid line): ability to “rule out” a disease correctly.
The test (and its sensitivity, which is its ability to detect cancer when present) is identically, but the prevalence of cancer
makes the test perform very differently at different institutions.
MOLECULAR TESTING

18. Pre-test risk of malignancy for each Bethesda category should be known to avoid incorrect
test interpretation and wrong surgical referral (routinally, it is almost impossible to know
the pretest malignancy risk for every single lab).
Therefore, before a molecular test is recommended to a patient, the pretest malignancy risk
should be determined and the assessment should also include other features to refine the
risk level such as:
MOLECULAR TESTING
Ferraz, Arch Endocrinol Metab 2018
A) family history of thyroid cancer.
B) patient’s history of radiation exposure.
C) cytological features (nuclear atypia, which increases the risk of malignancy).
D) ultrasound features (marked hypoechoic, microcalcifications, irregular margins).

19. MOLECULAR TESTING
Ferraz, Arch Endocrinol Metab 2018
Nikiforov was the first to report a gain in sensitivity (44% ->80%) and accuracy (from 93.3%
-> 97.4%) by analyzing a panel of BRAF, RAS, RET/PTC, and PAX8/PPARg mutations.
Based on this evidence, the first commercially available test named “miRInform” was
created (2009). It was later replaced and named (ThyGenX): BRAF, KRAS, HRAS, NRAS, and
chromosomal translocations resulting in RET/PTC1, RET/PTC3, and PAX8/PPARg fusions.
Mutations used as indipendent biomarkers their sensivity/specificity are too low to be
clinically relevant.
Prevalence of BRAF mutations in neoplastic indeterminate lesions is very low (4.6 %).

20. MOLECULAR TESTING
Ferraz, Arch Endocrinol Metab 2018
Next-generation sequencing (NGS) technologies is a DNA sequencing technology that
can be used to simultaneously sequence/discover genetic alterations in entire genomes
or be constrained to specific areas of interest.
ThyroSeq was the first targeted NGS panel customized for thyroid cancer.
In addition to the previous 7-Gene Panel, ThyroSeq includes: newly identified driver
mutations (PIK3CA, TP53, TSHR, PTEN, RET, AKT1, CTNNB1, TERT), gene fusions (involving
BRAF, RET, NTRK1, NTRK3, AKT1, PPARG, THADA).

21. MOLECULAR TESTING
Ferraz, Arch Endocrinol Metab 2018
An alternative to mutation testing: the analysis of differentially expressed genes
(definition of gene expression patterns).
The role of bioinformatics and the use of artificial models can create computer
algorithms and, thus, molecular classifiers.
Array technology is a powerful tool to assess the expression of a large number of
genes. The Afirma Gene Expression Classifier (GEC) embraces this approach by using
microarray technology on mRNA.
Using the same rationale as that of the GEC, after the first miR was discovered, miR
analyses gained an important place within the study of molecular markers.
ThyraMIR and RosettaGX Reveal are two miR panels that have been added to the market.

22. MOLECULAR TESTING
Ferraz, Arch Endocrinol Metab 2018

23. ThyroSeq v2 is a multigene test that is based on the targeted DNA and RNA next
generation sequencing analysis (NGS) of 56 genes (point mutations, small
insertions/deletions in 14 genes and 42 types of gene fusions) and expression levels of 16
genes.
Clinical validation of ThyroSeq v2 was reported in 3 single-institution studies of thyroid
nodules with indeterminate cytology and known surgical outcome.
Nikiforov Cancer 2014, Nikiforov Thyroid 2015
THYROSEQ V3

24. THYROSEQ V3
Mutations (BRAF V600E, TERT), are highly specific (100% risk of PTC). However, the
impact of preoperatively detecting RAS mutations or PAX8/PPARg fusions is still evolving.
False positive due to RAS mutations in benign lesion (gradual progression to malignancy?)
and some malignant lesions show no RAS mutations at all. Its identification in molecular
tests can be confusing.
ThyroSeq seems better in Bethesda IV than in Bethesda III.
Ferraz, Arch Endocrinol Metab 2018

25. MOLECULAR TESTS miRNA EXPRESSION
MicroRNAs are small noncoding RNAs that regulate gene expression by influencing the
stability and translation of mRNA.
Compared with mRNA, miRNAs are relatively stable and can be isolated from routinely
prepared formalinfixed histopathology or alcohol-fixed cytology samples, making them
a practical analyte for molecular diagnostics.
Two recent commercially available tests use miRNA expression:
ThyGenX/ThyraMIR
RosettaGX Reveal

26. THYGENX/THYRAMIR
ThyGenX is a targeted next-generation sequencing (NGS) test that assays for a small
number of mutations in 5 genes (BRAF, KRAS, HRAS, NRAS, and PIK3CA) and 3 gene
fusions (RET-PTC1, RET-PTC3, and PAX8-PPARG) associated with thyroid neoplasia.
The ThyraMIR miRNA expression classifier was introduced to improve ThyGenX panel.
ThyraMIR measures the expression levels of 10 miRNAs by PCR and uses a proprietary
algorithm to classify each nodule as having either a high-risk or low-risk miRNA profile
Labourier, J Clin End Metab 2015

27. ROSETTA GX REVEAL
RosettaGX Reveal analyzes miRNA expression patterns to classify indeterminate lesions
as having either a ‘‘benign’’ or ‘‘suspicious by miRNA profiling’’ result. Rosetta GX
Reveal’s test panel measures 24 miRNAs, 6 sequences of which closely overlap with
ThyraMIR’s panel of 10 miRNAs.
RosettaGX Reveal’s miRNA classifier was analytically validated on cellular material
recovered from direct cytology smears.
RosettaGX Reveal was clinically validated in a retrospective multicenter study of 189
cytologically indeterminate samples with histologic follow-up
Lithwick- Yanai, J Clin Pathol 2017

28. AFIRMA
The first published molecular test in NEJM (2012).
The most studied and clinically confirmed by post-validation studies.
Further improvements of Afirma: GEC -> GSC-> GSC X Atlas
The most advanced and studied molecular test available all over the world.
At the moment, the only one available in Italy for clinical use.

29. AFIRMA GEC
Alexander NEJM 2012, Alexander JCEM 2014.
The Afirma Gene Expression Classifier (GEC) is a microarray- based test that uses a
proprietary algorithm to risk stratify cytologically indeterminate nodules as having either
benign (GEC-B) or suspicious (GEC-S) messenger RNA (mRNA) expression profiles.

30. AFIRMA GEC
As a test optimized for high sensitivity and NPV, Afirma’s clinical utility is based on its
ability to help exclude malignancy in the setting of indeterminate cytology (rule out).
Gene expression cassettes screen for mRNA profiles associated with MTC (Afirma MTC test),
parathyroid tissue, and metastatic tumors.
Only samples that do not trigger these screening cassettes are analyzed by the main 142-
gene classifier; samples that are flagged by these screening cassettes are resulted as having
a suspicious

31. AFIRMA GEC
Alexander NEJM 2012, Alexander JCEM 2014.
Cytologically indeterminate nodules with GEC-B results (due to high NPV) can be
managed with clinical observation rather than diagnostic lobectomy in most cases, similar
to cytologically benign nodules.
The Afirma validation study remains the benchmark for assessing its test performance.
Nevertheless, the growing list of publications summarizing institutional experiences
with Afirma confirm its applicability in the real-world clinical setting.

32. AFIRMA GEC

33. AFIRMA GEC
Alexander NEJM 2012, Alexander JCEM 2014.
Afirma reduced the number of unnecessary surgeries.

34. AFIRMA GEC

35. AFIRMA GEC

36. AFIRMA GEC

37. AFIRMA GEC
Afirma GEC: high percentage of oxyphil lesions (Hurthle cells) classified as GES-
Sospicious (low specificity 12%).

38. AFIRMA GSC
Afirma Genomic Sequencing Classifier (GSC), combines classic RNA sequencing
with GEC microarray, with RNA sequencing that allows to extract as much genomic
information from the thyroid aspirate sample as possible and to identify genomic
information previously undetectable with other methods.
Afirma GSC, is the only test currently available that combines the sequencing of new
generation of RNA and analytical methods capable of detecting even small genomic
alterations not detectable by traditional methods (such as DNA-based sequencing).
Afirma GSC analyzes the RNA expression profiles of more than 1,000 genes: in
addition to gene expression, the presence of DNA variants, fusions, copy number
variants and other potentially predictive information of thyroid cancer is also
evaluated.
GSC compared to GEC, let to gain specificity and avoid histologically benign samples
to be classified as suspicious.

39. AFIRMA GSC

40. AFIRMA GSC

41. AFIRMA GSC

42. AFIRMA GSC

43. AFIRMA GSC

44. AFIRMA GSC X ATLAS

45. AFIRMA GSC X ATLAS

46. AFIRMA GSC X ATLAS

47. AFIRMA REPORT

48. AFIRMA REPORT

49. AFIRMA: OUR DATA
AFIRMA (N 18)
benign (12)
suspicious (5)
28 %
72 %
SOSPICIOUS:
1 -> thermoablation
1 -> follow-up
1 -> surgery: adenoma
2 (BRAF+) -> surgery (PTC)
BENIGN:
1 -> surgery: adenoma
40 %]

50. TECHNICAL CONSIDERATIONS
- For thyroid nodules larger than 10 mm.
- Complex procedure (import/export taxes, shipment costs, dollar exchange rate).
- Afirma cost is high but decreasing (3500->2400 €).
- Some of the health insurances cover the costs of the tests.
- Patients interested in molecular testing had to travel (USA, Canada, Israel, UAE).
- Now available in Italy (EndocrinologiaOggi, the first center in Europe).
- Application of Afirma is still far from daily clinical practice..
… but now it is a possibility, in Rome!

51. TAKE HOME MESSAGES
- Molecular testing are promising techniques.
- Each test was built in different way, with different objectives.
- Endocrinologists should know this different aspects.
- They represent the future but also the present.
- Patients start to know about molecular tests.
- They represent legal medical support.
- They are still expensive (but cost decreasing).
- Patients interested in molecular testing had to travel but not too far (now available in
Italy).

52. THANKS FOR YOUR ATTENTION…