Molecular Testing for Indeterminate Thyroid Nodules October 20, 2018
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
Follicular neoplasm Micro follicles
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
10-25% of FNA will lead to indeterminate thyroid nodules ATYPIA OF UNDETERMINED SIGNIFICANCE. Specimen consists of microfollicles, intranuclear inclusions and rare nuclear grooving.
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 6 18 10 40 Suspicious for malignancy 45 60 Malignant 94 96 0-32% 2-7% 6-48% 15-40% 53-87% 96-100% Edmund S. Cibas1 and Syed Z. Ali THYROID Volume 27, Number 11, 2017
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.?
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%)
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 10 40 Surgical lobectomy Suspicious for malignancy 45 60 Thyroidectomy Malignant 94 96 Thyroidectomy Baloch ZW et al. Diagn Cytopathol 2008;36:425 Cibas ES & Ali SZ. Am J Clin Pathol 2009;132:658
The challenge of managing Indeterminate thyroid nodules Indeterminate diagnosis Surgery Performed 10-40% Malignant 60-90%benign
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
The uncertainty of indeterminate thyroid FNAs can be resolved in 2 ways: Rule In High PPV Rule out High NPV
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.
Afirma Gene Expression Classifier (GEC) Molecular Testing for Thyroid FNA Nishino & Nikiforova Arch Pathol Lab Med Vol 142, April 2018
Afirma Gene Expression Classifier (GEC) Clinical Validation Study Cytological Dx n Sensitivity Specificity NPV PPV AUS 129 90% 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
Afirma Gene Expression Classifier (GEC) Post validation studies Alexander EK 2014, JCEM Harrell & Bimston, 2014. Endocr Prac McIver 2014, JCEM Lastra 2014, Cancer cytopathol Marti 2015 (MSK data), Annal of surg onc Brauner 2015. Thyroid Multiple 309 39% Single 56 50% Multiple 60 17% Single 132 44% Multiple 94 55% Multiple 71 13% High Sensitivity of 83-100 % High NPV 75-100% Low Specificity of 7-24% Low PPV of 14-57%
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
Published report of resected nodule with benign Afirma Gene Expression Classifier ( GEC)results
Update on Veracyte August 2017- Afirma Gene sequencing Classifier--- Higher specificity JAMA Surg. 2018;153(9):817-824. doi:10.1001/jamasurg.2018.1153 Published online May 23, 2018. PPV increased from 38-48% Specificity improved from 50-68% June 2018- Afirma Xpression Atlas- a panel of rich genomic content 761 DNA variants and 130 RNA fusions derived from 511 unique genes obtained through RNA
Multi- Gene Thyroseq Next Generation Sequencing Assay Molecular Testing for Thyroid FNA Nishino & Nikiforova Arch Pathol Lab Med Vol 142, April 2018
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%
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
Multi- Gene ThyroSeq Next Generation Sequencing Assay Cytolog ical Dx FN/ SFN n Sensit ivity Specif icity NPV PPV 143 90% 93% 96% 83% High specificity/ppv and high Sensitivity/NPV good rule in and rule out test
Multi- Gene Thyroseq Next Generation Sequencing Assay Cytologi cal Dx AUS/ FLUS n Sensitiv ity Specifici ty NPV PPV 465 91% 92% 97% 77% High specificity/ppv and high Sensitivity/NPV good rule in and rule out test
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
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
ThyGenX/ThyraMIR Molecular Testing for Thyroid FNA Nishino & Nikiforova Arch Pathol Lab Med Vol 142, April 2018
Cytological Dx n Sensitivity Specificity NPV PPV AUS 58 94% 80% 97% 68% FN/SFN 51 82% 91% 91% 82% total 109 89% 85% 94% 74% High specificity/ppv and high Sensitivity/NPV good rule in and rule out test
(J Clin Endocrinol Metab 103: 2261 2268, 2018) GEC ThyroSeq v2 Further studies are required for comparison with other available molecular diagnostics and for newer tests as they are developed
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
Ultrasound Molecular testing Cytology Surgery observation
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
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?????????
Pre-Test probability of cancer in URMC indeterminate thyroid nodules based on ultrasonographic and cytologic parameters.
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 1.90 0.98 3.66 0.05 Hypervascularity 1.71 0.94 3.10 0.08 Variable Odds ratio Lower 95% CL Upper 95% CL P-value Nuclear Atypia 2.12 1.17 3.84 0.01
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 4.93 1.30 18.73 0.019 Microcalcification and Nuclear Atypia 36.14 3.9 331.11 0.002 Hypervascularity and Nuclear Atypia 3.65 1.27 10.51 0.017 Microcalcification, Hypervascularity and Nuclear Atypia 6.57 1.80 23.98 0.004
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
Patient 1: Our Probability of cancer was low- Afirma helped patient avoid surgery
Patient 2: Our Probability of cancer was high- Afirma did not help to change the management in this patient
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
Thank you!!