Molecular Testing for Indeterminate Thyroid Nodules. October 20, 2018
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1 Molecular Testing for Indeterminate Thyroid Nodules October 20, 2018
2 Patient 1: Left 1.0 cm AP x 1.6 cm transverse x 2.1 cm in length Well defined Isoechoic heterogeneous No calcification Grade 3 Vascularity ATA RISK- low to intermediate
3 Follicular neoplasm Micro follicles
4 Patient 2: Ill defined Hypoechoic heterogeneous +Macro calcification Grade 2 Vascularity ATA RISK- high risk Right 1.2 x 1.2 x 1.8 cm
5 10-25% of FNA will lead to indeterminate thyroid nodules ATYPIA OF UNDETERMINED SIGNIFICANCE. Specimen consists of microfollicles, intranuclear inclusions and rare nuclear grooving.
6 The 2017 Bethesda system for reporting thyroid cytopathology Diagnostic category Bathesda malignancy Risk Reported Malignancy risk Non-diagnostic or unsatisfactory 5 10 Benign 0 3 Atypia of undetermined significance Higher or interobserver follicular lesion of Variability undetermined significance Follicular neoplasm or suspicious for a follicular neoplasm Suspicious for malignancy Malignant % 2-7% 6-48% 15-40% 53-87% % Edmund S. Cibas1 and Syed Z. Ali THYROID Volume 27, Number 11, 2017
7 This raised 2 important questions 1- What's in the incidence of indeterminate thyroid nodules at URMC? 2- What is the risk of cancer in indeterminate thyroid nodules at our institution.?
8 URMC data on the prevalence of Indeterminate thyroid nodules and risk of malignancy Total number of thyroid nodules FNA n=5,080 Non-diagnostic :5.4% n=278 Thyroid nodules with cytological diagnosis n=4802 Benign: 86%(n= 4119) Malignant : 6.9% (n=333) IDN: 7.2 % (n=350) Excluded n=105 IDN included in study 245 Benign 141 ( 57%) Malignant 104 ( 42%)
9 The Usual Management Diagnostic category Bathesda malignancy Risk Management Non-diagnostic or unsatisfactory 5 10 Repeat FNA Benign 0 3 Clinical follow up Atypia of undetermined significance or follicular lesion of undetermined significance Follicular neoplasm or suspicious for a follicular neoplasm 6 18 Repeat FNA Surgical lobectomy Suspicious for malignancy Thyroidectomy Malignant Thyroidectomy Baloch ZW et al. Diagn Cytopathol 2008;36:425 Cibas ES & Ali SZ. Am J Clin Pathol 2009;132:658
10 The challenge of managing Indeterminate thyroid nodules Indeterminate diagnosis Surgery Performed 10-40% Malignant 60-90%benign
11 So the question is 1- What is the chance that a person with positive test truly has the disease? 2- What is the chance that a person with negative test is disease free? Test Positive Test Negative Cancer True Positive ( TP) False Negative ( FN) Total cancer TP+FN Benign False Positive (FN) True negative (TN) Total Benign TN+FP Sensitivity: Ability of test to identify disease Specificity: Ability of test to identify those with out disease PPV: The probability of presenting the disease when the test is positive Thus to predict malignancy, it will need to have a high PPV NPV: The probability of being free of disease when the test is negative Thus to predict benign nodules, it will require a high NPV
12 The uncertainty of indeterminate thyroid FNAs can be resolved in 2 ways: Rule In High PPV Rule out High NPV
13 The Molecular tests - The Afirma Gene Expression Classifier (GEC) - ThyroSeq v2 - ThyGenX/ThyraMIR The goals of ancillary molecular testing for thyroid cytology include (1) Avoidance of unnecessary surgery for benign nodules (2) Distinguishing high-risk cancers that merit total thyroidectomy from premalignant or low/intermediate-risk nodules for which lobectomy may be the preferred initial surgical step.
14 Afirma Gene Expression Classifier (GEC) Molecular Testing for Thyroid FNA Nishino & Nikiforova Arch Pathol Lab Med Vol 142, April 2018
15 Afirma Gene Expression Classifier (GEC) Clinical Validation Study Cytological Dx n Sensitivity Specificity NPV PPV AUS % 53% 95% 38% FN/SFN 81 90% 49% 94% 37% Susp 55 94% 52% 85% 76% High sensitivity/npv, low specificity/ppv good rule out test
16 Afirma Gene Expression Classifier (GEC) Post validation studies Alexander EK 2014, JCEM Harrell & Bimston, Endocr Prac McIver 2014, JCEM Lastra 2014, Cancer cytopathol Marti 2015 (MSK data), Annal of surg onc Brauner Thyroid Multiple % Single 56 50% Multiple 60 17% Single % Multiple 94 55% Multiple 71 13% High Sensitivity of % High NPV % Low Specificity of 7-24% Low PPV of 14-57%
17 Clinical Utility Afirma Gene Expression Classifier (GEC) Among the Afirma GEC benign patients, only 122 of the 1211 patients (10%) were operated, demonstrating a dramatic reduction in surgery compared to the 73% historical rate of surgery when Afirma was not used All cytology indeterminate with out Afirma GEC Afirma GEC benign
18 Published report of resected nodule with benign Afirma Gene Expression Classifier ( GEC)results
19 Update on Veracyte August Afirma Gene sequencing Classifier--- Higher specificity JAMA Surg. 2018;153(9): doi: /jamasurg Published online May 23, PPV increased from 38-48% Specificity improved from 50-68% June Afirma Xpression Atlas- a panel of rich genomic content 761 DNA variants and 130 RNA fusions derived from 511 unique genes obtained through RNA
20 Multi- Gene Thyroseq Next Generation Sequencing Assay Molecular Testing for Thyroid FNA Nishino & Nikiforova Arch Pathol Lab Med Vol 142, April 2018
21 Multi- Gene Thyroseq Next Generation Sequencing Assay Gene Mutations BRAF NRAS HRAS KRAS PIK3C A PTEN TSHR AKT1 TP53 GNAS CTNNB1 RET LOW Sensitivity: 50-65% HIGH Specificity: >80%
22 ThyroSeq V2 NGS Mutation panel 14 genes for mutation, 42 fusion types, 16 genes for expression Gene Mutations Gene Fusions Gene Expressions BRAF TSHR RET PGK1 NRAS AKT1 PPARG KRT7 HRAS TP53 NTRK1 TG KRAS GNAS NTRK3 TTF1 PIK3CA CTNNB1 BRAF NIS PTEN RET ALK Calcitonin TERT EIF1AX Other PTH KRT20 Other
23 Multi- Gene ThyroSeq Next Generation Sequencing Assay Cytolog ical Dx FN/ SFN n Sensit ivity Specif icity NPV PPV % 93% 96% 83% High specificity/ppv and high Sensitivity/NPV good rule in and rule out test
24 Multi- Gene Thyroseq Next Generation Sequencing Assay Cytologi cal Dx AUS/ FLUS n Sensitiv ity Specifici ty NPV PPV % 92% 97% 77% High specificity/ppv and high Sensitivity/NPV good rule in and rule out test
25 Moffitt Cancer Center In 102 resected ITNs, ThyroSeq v2 performance was as follows: sensitivity 70% specificity 77% PPV 42% NPV 91% Mutations in RAS were the most prevalent Mount Sinai 156 ITN NPV >95 PPV 22% Mutations in RAS were the most prevalent
26 Update on Thyroseq ThyroSeq v3 is a DNA- and RNA-based next-generation sequencing assay that analyzes 112 genes Double-blind multicenter international study 234 patients (257 nodules) with Bethesda III, IV cytology and surgery NPV > 95% PPV 64-68% Steward JAMA Oncology 2018 in press
27 ThyGenX/ThyraMIR Molecular Testing for Thyroid FNA Nishino & Nikiforova Arch Pathol Lab Med Vol 142, April 2018
28 Cytological Dx n Sensitivity Specificity NPV PPV AUS 58 94% 80% 97% 68% FN/SFN 51 82% 91% 91% 82% total % 85% 94% 74% High specificity/ppv and high Sensitivity/NPV good rule in and rule out test
29 (J Clin Endocrinol Metab 103: , 2018) GEC ThyroSeq v2 Further studies are required for comparison with other available molecular diagnostics and for newer tests as they are developed
30 Afirma ThyroSeq ThygenX/ThyraMIR Company Veracyte University of Pittsburg Med Ctr, via CBL path Methodology mrna Microarray analysis Next Generation sequencing to detect mutations and rearrangements Strengths High The NPV overall mean cost High of NPV/PPV outpatient Validated in blinded thyroidectomy was $5617, with a multicenter prospective trial Limitations Low PPV New test with limited real world experience, overnight observation Histology diagnosis not blinded to prior molecular testing results in validation study Cost mean cost of same-day surgery of $4642 compared with $6101 for 4875$ for AFIRMA GEC and MTC Interspace Diagnostics ThyGenX:Multiplex PCR to detect mutations and rearrangements ThyraMIR: Microarray expression analysis High NPV/PPV Validated in blinded prospective multicenter study New test with limited real world experience 3200$ 1675$ for THYGenX and 3300 for ThraMIR
31 Ultrasound Molecular testing Cytology Surgery observation
32 Indeterminate thyroid nodules Pretest probability of thyroid cancer If the pre test Cancer Risk is Lower based on patient history, ultrasonographic and cytologic features Can Patient AVOID surgery? Repeat FNA or use molecular test with a validated high NPV Afirma ThyroSeq
33 Indeterminate thyroid nodules Pretest probability of thyroid cancer If the pre test Cancer Risk is higher Hence the NPV may not be high as reported studies. Patient likely needs surgery Does Molecular test help in extent of surgery?????????
34 Pre-Test probability of cancer in URMC indeterminate thyroid nodules based on ultrasonographic and cytologic parameters.
35 Multivariate analysis of thyroid ultra-sonographic features and cytological parameters associated with malignancy Variable Odds ratio Lower 95% CL Upper 95% CL P-value Microcalcifications Hypervascularity Variable Odds ratio Lower 95% CL Upper 95% CL P-value Nuclear Atypia
36 Multivariate Analysis with cytological parameter and thyroid ultra-sonographic features associated with malignancy Variables Odds ratio Lower 95% CL Upper 95% CL P-value Microcalcification and Hypervascularity Microcalcification and Nuclear Atypia Hypervascularity and Nuclear Atypia Microcalcification, Hypervascularity and Nuclear Atypia
37 Coming back to patients Probability of cancer Risk Patient #1 Patient #2 Ultrasonographic features: low risk; Ultrasonographic features: high risk; well defined solid heterogamous solid hypoechoic, microcalcifications nodule and increase internal vascularity Cytologic features : architectural features in the form of microfollicles Cytologic features : Nuclear features in the form of grooving and inclusion The probability of cancer is low based on ultrasonographic and cytologic features The probability of cancer is high based on ultrasonographic and cytologic features
38 Patient 1: Our Probability of cancer was low- Afirma helped patient avoid surgery
39 Patient 2: Our Probability of cancer was high- Afirma did not help to change the management in this patient
40 In a Nut shell - Know your Institution pathologist---most important Risk based approach - Know your Institution prevalence of indeterminate nodules and risk of Patient History cancer. Ultrasonographic features Cytologic parameters - Choose the test based Molecular on the testing probability of cancer Surgery Observation Patient preference Cost effectiveness
41 Thank you!!
42
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