Molecular biopathology of thyroid tumors

Molecular biopathology of thyroid tumors Philippe Vielh MD, PhD, FIAC Director of Cytopathology Deputy Director of Anatomic Pathology National Health Laboratory of Luxembourg Past President of the International Academy of Cytology philippe.vielh@lns.etat.lu Conflict of interest: no disclosure

OUTLINES Background Current evaluation of thyroid gland lesions Carcinogenesis model(s) Is it useful to assess genetic alterations on FNAC? Conclusions

BACKGROUND Thyroid nodules are frequent (2-30%) Carcinomas are rare (5%) and most are differentiated L. Da Vinci, 1500 Most differentiated carcinomas are of good prognosis Goya, 1804

Lazzeri D, et al. Thyroid (2015);25:559 562

Prevalence of thyroid nodules vs age Mazzaferri EL, N Engl J Med,1993,328:553-559

DTC: Incidence and extent of disease Increasing incidence of cancers (3%-6%/year for 30 years). Attributed mainly to improved screening T3 T4: stable incidence ~10% Distant metastases:<10% of patients with clinical differentiated TC. Cancer is present in only 5% of all thyroid tumors: diagnosis is first based on FNAC Sassolas et al. Eur J Endocrinol 2009;160:71

Furuya-KanamoriL, et al. J ClinOncol(2016) 34

Thyroid-Cancer Incidence and Related Mortality in South Korea, 1993 2011. Data on incidence are from the Cancer Incidence Database, Korean Central Cancer Registry; data on mortality are from the Cause of Death Database, Statistics Korea. All data are age-adjusted to the South Korean standard population. AhnHS, et al. N EnglJ Med (2014) 371;19:1765-67

Penetration of Thyroid-Cancer Screening (2008 2009) and Incidence of Thyroid Cancer (2009) in the 16 Administrative Regions of South Korea. Data on thyroidcancer screening are from the Korean Community Health Survey Database, Korea Centers for Disease Control and Prevention; data on incidence are from the Cancer Incidence Database, Korean Central Cancer Registry. AhnHS, et al. N EnglJ Med (2014) 371;19:1765-67

CURRENT EVALUATION OF THYROID GLAND LESIONS Clinical examination Ultrasonography (+/- scintigraphy) Biochemical dosages (TSH, calcitonine) Fine-needle aspiration cytology (FNAC) Thyroid Lymph-node compartment of the neck Histopathological evaluation if needed

Lymph-node compartments of the neck Figure 2: Lymph-node compartments of the neck Level VI is also known as the central neck compartment and includes the prelaryngeal, pretracheal, and paratracheal lymph nodes. Reproduced from Cooper and colleagues, by permission of the American Thyroid Association. Lancet 2013; 381: 1046 57

ATA recurrence risk stratification system Lancet 2013; 381: 1046 57

CARCINOGENESIS MODELS Nikiforova M. Expert Rev. Mol. Diagn. 2008;8:83

Carcinogenesis models Kondo T. Nature Rev Cancer 2006;6:292

Early genetic events Kondo T. Nature Rev Cancer 2006;6:292

Gene mutations in thyroid tumors Xing M, Nat Rev Cancer 2013,13:184-199

Model of multistep carcinogenesis Pallante, P. et al. Nat. Rev. Endocrinol. 2014,10;88 101 Figure 1 Classification of human thyroid carcinomas and subtype-specific genetic alterations. The thyroid gland contains follicular cells and C cells (also known as parafollicular cells). The vast majority of benign thyroid tumours are FTAs that often carry mutations in the RAS gene. MTCs derive from C cells and often have mutations in the proto-oncogene RET. RAS mutations and PAX8 PPARG translocation have been associated with FTCs. Follicular-cell-derived carcinomas can be subdivided into three categories: well-differentiated, poorly differentiated and undifferentiated thyroid carcinomas or ATCs. Well-differentiated thyroid carcinomas can be further classified under the PTC and FTC subtypes. Rearrangements lead to the formation of the RET/PTC oncogene, which is often found in PTC. Rearrangements involving NTRK1 together with point mutations in RAS and BRAF are also involved in the development of PTC. Deregulation of the PI3K AKT pathway through mutations affecting PIK3CA, PTEN, AKT1, CTNNB1, as well as loss of the tumour suppressor retinoblastoma (RB), is involved in the loss of differentiation, thereby leading from PTC and FTC to undifferentiated carcinomas. P53 mutations lead to a completely undifferentiated state in ATC. Abbreviations: ATC, anaplastic thyroid carcinomas; FTA, follicular thyroid adenomas; FTC, follicular thyroid carcinomas; MTC, medullary thyroid carcinoma; PTC, papillary thyroid carcinoma.

Bible KC, et al. Nat Rev Clin Oncol (2016);13:403 416

Raue F, et al. Clin Cancer Res (2016);22(20):5012-5021

Models of carcinogenesis Xing M, Nat Rev Cancer 2013,13:184-199 Figure 3 Model of the progression of thyroid tumorigenesis driven by the MAPK and PI3K AKT pathways. Activation of the MAPK pathway by genetic alterations, such as the BRAFV600E mutation, primarily drives the development of papillary thyroid cancer (PTC) from follicular thyroid cells. By contrast, activation of the PI3K AKT pathway by genetic alterations, such as mutations in RAS, PTEN and PIK3CA, primarily drives the development of follicular thyroid adenoma (FTA) and follicular thyroid cancer (FTC) from follicular thyroid cells. Conversion from FTA to FTC is largely due to increasing activation of the PI3K AKT pathway. As genetic alterations accumulate and intensify the signalling of each of the two pathways, PTC and FTC can progress to poorly differentiated thyroid cancer (PDTC). When both pathways are fully activated through accumulated genetic alterations, conversion from PDTC to anaplastic thyroid cancer (ATC) is strongly facilitated. It is also possible that PDTC and ATC can both develop de novo directly from follicular thyroid cells, and that ATC can develop from PTC or FTC if appropriate genetic alterations occur. Many secondary molecular alterations also progressively accumulate and synergize with the two pathways in driving the progression of thyroid tumorigenesis, as discussed in the text (not shown in the figure). The increasing number of vertical arrows and colour intensity of the ovals symbolize the increasing genetic alterations and signalling of the two pathways as thyroid tumorigenesis progresses. The figure is modified from REF. 33 (2007) American Association for Cancer Research.

Clinical management Pathologic differentiation Well-differentiated: follicular, papillary, medullary Others: poorly-differentiated, undifferentiated Tumour stage Early Others: advanced, metastatic, persistent, recurrent

Clinicopathological features in adults Xing M, Nature Rev Cancer 2013;184:199

Clinicopathological features in adult Kondo T. Nature Rev Cancer 2006;6:292

Clinicopathological features in childhood Yamashita S. Nature Clin Practice Endocrinol Met 2007;3:422

Treatment Primary surgery + adjuvant treatment for well-differentiated tumours at an early phase Targeted therapy for poorly or undifferentiated carcinomas as well as in - persistent, recurrent - advanced or metastatic tumours

Cell type and risk of cancer Santoro M. Nature Clin Practice Endocrinol Metabol 2006;2:42

Consequences of molecular lesions Santoro M. Nature Clin Practice Endocrinol Metabol 2006;2:42

IS IT USEFULNESS TO ASSESS GENETIC ALTERATIONS ON FNAC? At diagnosis? in well-differentiated tumours detected at an early stage: papillary; medullary; follicular As predictor to treatment? for targeted therapy: poorly-differentiated or undifferentiated carcinomas and advanced, metastatic, persistent or recurrent tumours

At diagnosis? Adults Children & adolescents Filetti S. Nature Clin Practice Endocrinol Met 2006;2:384 Dinauer C. Curr Opin Oncol 2008;20:59

Prevalence in adults Nikiforov Y. Modern Pathol 2008;21:S37

RET gene rearrangements in thyroid papillary carcinoma Nikiforova M. Expert Rev. Mol. Diagn. 2008;8:83

Prevalence of genetic alt. in children vs adult thyroid papillary ca. Yamashita S. Nature Clin Practice Endocrinol Met 2007;3:422

Adult vs children genetic alterations in thyroid papillary carcinoma Yamashita S. Nature Clin Practice Endocrinol Met 2007;3:422

Papillary carcinoma tumorigenesis Sobrinho-Simoes M. Virchows Arch 2005;447:787

Follicular variant of papillary carcinoma tumorigenesis Sobrinho-Simoes M. Virchows Arch 2005;447:787

Molecular pathways of various thyroid papillary carcinoma Nikiforova M. Expert Rev. Mol. Diagn. 2008;8:83

Follicular lesions tumorigenesis Sobrinho-Simoes M. Virchows Arch 2005;447:787

(PAX8-)PPARgamma rearrangement

Molecular pathways of various thyroid follicular tumours Nikiforova M. Expert Rev. Mol. Diagn. 2008;8:83

Diff. diagnosis of follicular lesions Diagn Cytopathol 2008;36:438

Dual goal of thyroid nodule evaluation Yip L www.co-oncology.com 2014,26:8-13 1) Accurately exclude thyroid malignancy High negative predictive value (NPV) High sensitivity 2) Effectively optimize initial surgical management High positive predictive value (PPV) High specificity

Molecular tests on FNAC Hodak SP, Thyroid 2013, 23, 131-134 VERACYTE Afirma Commercially available (Multi) gene expression classifier (GEC) proprietary «Rule out» test (high NPV) Identifying lesions highly likely to be benign (therefore obviating the need for diagnostic surgery) or suspicious (!) Alexander EK (NEJM 2012,367:705-715) with 265 cytologically indeterminate nodules Rate of histologic malignancy = 32% NPV = 93% (PPV = 47%) Sensitivity = 92% i.e. 8% (7/85) of cancers uncorrectly identified as benign lesions Specificity = 52% i.e. 48% of benign nodules uncorrectly identified as «suspicious» Percentage of cancers missed in: AUS/FLUS = 10% FN/SFN = 10% Suspicious for malignancy = 6% Cost-effectiveness study (3200US dollars): Li H (JCEM 2011,96:E1719-E1726) In indeterminate FNAC, the unreliable NPV of 89% in nodules with suspicious cytologic results and the overall high false positive rate prevents the use of this test to guide appropriate surgical treatment.

Molecular tests on FNAC Hodak SP, Thyroid 2013, 23, 131-134 ASURAGEN mirinform thyroid panel Commercially available «rule in» method (high PPV) identifying lesions highly associated with malignant histology Including point mutations : BRAF (V600E and K601E) and (N-, K-, and H-RAS hotspots in codons 12, 13 and 61) and rearrangements (RET/PTC1 and 3 and PAX8/PPARg) One of the non overlapping mutations is identified in up to 75% of papillary carcinomas and 70% of follicular carcinomas Nikiforov YE (JCEM 2011,96:3390-3397) with 513 histologically correlated (rate of histologic malignancy = 24%) lesions. For indeterminate cytology: PPV = 89% (NPV = 89%) Sensitivity = 61% Specificity = 98% Percentage of cancers missed in: AUS/FLUS = 37% FN/SFN = 43% Suspicious for malignancy = 32% In indeterminate FNAC, the high PPV and high specificity of the panel accurately identify nodules with a high risk of cancer but negative panel testing results still have a 14% risk of cancer Cost-effectiveness study (2250 US dollars): Yip L (JCEM 2012,97:E1905-1912)

Molecular tests on FNAC Yip L www.co-oncology.com 2014,26:8-13

Molecular tests on FNAC (AUS/FLUS) to guide surgery Gomberawalla A, www.co-oncology.com 2014,26:14-21 FIGURE 1. Proposed algorithm using molecular testing to guide decision-making in patients with fine-needle aspiration biopsies in the atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) category. NPV, negative predictive value; PPV, positive predictive value.

Molecular tests on FNAC (FN/SFN) to guide surgery Gomberawalla A, www.co-oncology.com 2014,26:14-21 FIGURE 2. Proposed algorithm using molecular testing to guide decision-making in patients with fine-needle aspiration biopsies in the follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) category. NPV, negative predictive value; PPV, positive predictive value.

Molecular tests on FNAC (SMC) to guide surgery Gomberawalla A, www.co-oncology.com 2014,26:14-21 FIGURE 3. Proposed algorithm using molecular testing to guide decision-making in patients with fine-needle aspiration biopsies in the suspicious for malignant cells (SMCs) category. NPV, negative predictive value; PPV, positive predictive value.

Molecular tests on FNAC for management of thyroid nodules Xing M, Lancet 2013,381:1058-1069 Figure 3: Algorithm for management of thyroid nodules on the basis of FNAB and molecular marker tests Depending on the cytology categories, molecular tests with high sensitivity and NPV (eg, gene expression classifi er) or high specifi city and PPV (eg, BRAF mutation) are chosen. Extent of surgery should be decided on the basis of the combined assessment of clinical, imaging, cytological, and molecular marker data. FNAB=fi ne needle aspiration biopsy. AUS/FLUS=atypia of undetermined signifi cance/follicular lesion of undetermined signifi cance. FN/SFN=follicular neoplasm/suspicious for follicular neoplasm. PTC=papillary thyroid cancer. NPV=negative predictive value. PPV=positive predictive value. Tx=total/near total thyroidectomy. LND=lymph node dissection.

Molecular tests on FNAC Yip L, www.co-oncology.com 2014,26:8-13 TARGETED NEXT GENERATION SEQUENCING PANEL (ThyroSeq) Nikiforova MN, JCEM 2013,98:E1852-1860 o Expansion of the gene hotspots screened o Increase the number of gene hotspots evaluable with a low quantity of DNA o Improvement of the sensitivity of mutant allele detection EXOME NEXT GENERATION SEQUENCING

As therapeutic predictor?

MAPK and other pathways in thyroid tumors Figure 1 The MAPK and related pathways in thyroid cancer. Shown in the middle of the figure is the classical MAPK pathway leading from an extracellular mitogenic stimulus that activates a receptor tyrosine kinase (RTK) in the cell membrane, to RAS, RAF (shown as BRAF-V600E), MEK and ERK. ERK is activated by phosphorylation (P) and enters the nucleus where it upregulates tumour-promoting genes and downregulates tumour suppressor genes and thyroid iodide-handling genes. On the left side of the figure is the nuclear factor-κb (NF-κB) pathway, in which extracellular stimuli activate the pathway by acting on receptors in the cell membrane, leading to activation of the inhibitor of κb (IκB) kinase (IKK), resulting in the phosphorylation of IκB. Phosphorylated IκB becomes dissociated from NF-κB, which is normally bound with IκB in a complex and sequestered in the cytoplasm. Phosphorylated IκB undergoes ubiquitylation and proteasomal degradation. Free NF-κB then enters the nucleus to promote the expression of tumour-promoting genes. Through an undefined mechanism that is independent of MEK signalling, BRAF-V600E promotes the phosphorylation of IκB and the release of NF-κB, thus activating the NF-κB pathway. Shown on the right side of the figure is the RASSF1 mammalian STE20-like protein kinase 1 (MST1) forkhead box O3 (FOXO3) pathway. Activated by extracellular pro-apoptotic stimuli through membrane receptors, RASSF1A activates MST1. Activated MST1 then phosphorylates FOXO3 on Ser207. The resulting phosphorylated FOXO3 becomes dissociated from 14-3-3 proteins in the cytoplasm. 14-3-3 proteins undergo proteasomal degradation, and phosphorylated FOXO3 enters the nucleus to promote the expression of proapoptotic genes in the FOXO pathway. BRAF-V600E directly interacts with and inhibits MST1 and prevents its activation by RASSF1A, thereby suppressing the proapoptotic signalling of the FOXO3 pathway. The downward arrow for the FOXO activities shown in the nucleus indicates this negative effect of BRAF-V600E on proapoptotic genes, which are normally upregulated by the RASSF1A MST1 FOXO3 pathway. The triple independent coupling of BRAF-V600E to the pathways shown here represents a unique and powerful mechanism of thyroid tumorigenesis driven by BRAF-V600E. DAPK1, death-associated protein kinase 1; HIF1A, hypoxia-inducible factor 1α; MMP, matrix metalloproteinase; NIS, sodium iodide symporter; TGFB1, transforming growth factor β1; TIMP3, tissue inhibitor of metalloproteinases 3; TPO, thyroid peroxidase; TSHR, thyroid-stimulating hormone receptor; TSP1, thrombospondin 1; UPA, urokinase plasminogen activator; UPAR, urokinase plasminogen activator receptor; VEGFA, vascular endothelial growth factor A. Xing M, Nat Rev Cancer 2013,13:184-199

MAPK and PI3K-mTOR pathways in thyroid tumors Xing M, Lancet 2013; 381: 1058 69 Figure 1: MAPK and PI3K-AKT-MTOR pathways genetic alterations and therapeutic targets in thyroid cancer Right side shows the MAPK pathway; left side shows the PI3K-AKT- MTOR pathway. The two classic signalling pathways are coupled to the receptor thyrosine kinase (RTK) at the cell membrane which transduces extracellular growth signals into intracellular signalling downstream of the two pathways. RAS can couple the signalling from RTK to both pathways. PTEN terminates the PI3K signalling. Genetic RTK amplifi cations are common. Common activating mutations in the MAPK pathway include RET-PTC rearrangement, RAS mutation, and BRAF mutation. Common genetic alterations in the PI3K pathway include RAS mutation, PTEN mutation or deletion, PIK3CA mutation or amplifi cation, and AKT1 mutation. The two pathways, driven by these genetic alterations, have a fundamental role in thyroid tumorigenesis. Amplifi cations of RTK genes are also common. *Denotes therapeutic targets in the two pathways that are currently being actively tested clinically.

Strategies for targeting RET tyrosine kinase receptor activation Nikiforova M. Expert Rev Anticancer Ther 2008;8:625

Small molecule inhibitors of RET in current clinical trials for MTC Schlumberger M. Nat Clin Pract Endocrin Met 2008;4:22

Therapeutic stratification in anaplastic carcinoma Immunohistochemical detection of: EGFr : 58% PDGFrbeta : 16% HER2 : 16%

Cell-cycle regulation Kondo T. Nature Rev Cancer 2006;6:292

Cell-cell and cell-matrix interactions Kondo T. Nature Rev Cancer 2006;6:292

Anti-Vasculo-Endothelial-Receptors Kerbel R. N Engl J Med 2008;358:2039

Multitargeted therapy Deshpande H. Curr Op Oncol 2008;20:19

Bible KC, et al. Nat Rev Clin Oncol(2016);13:403-16

Raue F, et al. Clin Cancer Res (2016);22(20):5012-5021

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CONCLUSIONS FNAC is currently the best approach for characterizing thyroid nodules; common language suggested by the Bethesda consensus meeting; still a morphological «grey zone» which may be the niche for molecular tests also helpful to identify prognostic biomarkers as well as targets for precision medicine.

Grazie!

Physiology Kondo T. Nature Rev Cancer 2006;6:292

Oncocytic tumours tumorigenesis Sobrinho-Simoes M. Virchows Arch 2005;447:787