This contractor will cover molecular diagnostic tests for use in a beneficiary
with an indeterminate or suspicious thyroid nodule when all the following
criteria are met:

  * The patient: 
    * Has not been tested with the same or similar assay for the same clinical indication AND: 
      * Has an indeterminate thyroid nodule as defined by Bethesda categories III-IV OR
      * Has a Bethesda category V nodule for which molecular testing may aid in further stratifying the type of malignancy.
  * The results of the test will be used to aid in surgical decision making after a consideration of clinical, radiographic and cytologic features.
  * The beneficiary is within the population and has the indication for which the test was developed. The laboratory providing the test is responsible for clearly indicating to treating clinicians the population and indication for test use.
  * The test demonstrates analytical validity, including both analytical and clinical validation, on a cohort of patients appropriate for its intended use. If the test relies on an algorithm, the algorithm must be validated in a cohort that is not a development cohort for the algorithm.
  * The test has demonstrated clinical validity and utility in peer-reviewed, published literature, establishing a clear and significant biological/molecular basis for stratifying patients and subsequently selecting (either positively or negatively) a clinical management decision in a clearly defined population.
  * The test successfully completes a technical assessment that ensures the test is reasonable and necessary as described above.
  * The performance characteristics of the test have been demonstrated to be as good or better than currently covered services.

NOTE: Next Generation Sequencing (NGS) performed to identify genetic variants
in samples classified as malignant is not within the scope of this policy but
may fall under other established policies.

Thyroid cancer (TC) is the most common endocrine malignancy, consisting of
nearly 3% of all newly diagnosed cancer cases in the United States each year.1
Greater than 70% of those cases are women, representing the fifth most
diagnosed malignancy in females. It is the second most common cancer among
Hispanic and Asian/Pacific Islander women in the US, who also have the highest
mortality rates.2 Differentiated thyroid carcinoma is the most common form,
accounting for around 90% of all cases and includes papillary thyroid
carcinoma (PTC), follicular carcinoma (FC) and Hurthle cell carcinoma
(HTC).3,4 Medullary thyroid carcinoma (MTC), a rare neuroendocrine tumor that
arises from the neural crest-derived parafollicular calcitonin-secreting
thyroid C cells, represents 4% of all TC.5 Anaplastic thyroid carcinoma (ATC)
is the most aggressive thyroid tumor and while only around 1-2% of all TC,
accounts for the majority of TC death.6

The diagnosis of TC in the United States has tripled over the last 25 years.1
Several studies attribute the significant increase to overdiagnosis of small
indolent tumors that would otherwise not cause symptoms or require treatment
with a majority of the increase being explained by PTC tumors 2 cm or
smaller.7-11 In fact, recent studies suggest the incidence rate of thyroid
cancer stabilized between 2013-2016 and declined between 2016-2018.12,13 This
stabilization followed by decline has been postulated to be a result of
changes in practice patterns and reclassification of some cancer types. In
2016, the Endocrine Pathology Society working group reported clinical outcomes
and refined the diagnostic criteria for encapsulated follicular variant of
papillary thyroid carcinoma (EFVPC) and proposed replacing the term with non-
invasive follicular thyroid neoplasm with papillary-like nucleus features
(NIFTP) to describe these tumors more accurately.14 The American Thyroid
Association (ATA) recommended this terminology change in 2017.15 This led to
the reclassification of approximately 10-20% of thyroid tumors from malignant
to benign.14 In addition, guidelines for the management of thyroid nodules
have become increasingly more conservative regarding size thresholds for
nodule biopsy and discourage biopsy for nodules < 1 cm.16,17 However, some
studies have reported a true increase in advanced-stage and larger PTC tumors
as well as incidence-based mortality that cannot be explained by overdiagnosis
and suggest that lifestyle-related factors such as obesity may be
contributory.10,18 Also, there continue to be disparities in diagnosis and
treatment of TC in patients based on race, ethnicity and socioeconomic status
with patients from minority backgrounds more likely to present with larger
tumors, and distant metastases than white patients.2,19,20 However, a recent
report from Ginzberg et al. suggests that the updated ATA guidelines
ameliorated some of these disparities.21

TC almost exclusively presents as thyroid nodules, occurring in 7-15% of cases
depending on gender, age, radiation exposure, family history and other
factors.16 However, thyroid nodules are very common; most are asymptomatic and
benign and do not require monitoring, treatment, or evaluation. In fact, over
60% of the population will have a thyroid nodule by the time they are over the
age of 65.16 Therefore, it is important to distinguish between benign and
malignant nodules for patients to receive appropriate treatment and prevent
unnecessary surgery.

The malignancy potential of a thyroid nodule is determined through a
multimodality manner including physical exam, personal and family history,
radiographic assessment, and fine needle aspiration (FNA) biopsy. FNA biopsies
are the procedure of choice when evaluating clinically suspicious thyroid
nodules and every year more than 500,000 of this minimally invasive procedure
are performed.22 The results of FNA biopsies are reported using the Bethesda
System for Reporting Thyroid Cytopathology (TBSRTC).23 This system was
established to provide consensus recommendations for diagnostic categories for
FNA specimens with a goal of standardizing classification and reporting across
health care providers. It includes recommendations on sample adequacy,
malignancy risk, report layout and management and has been widely adopted. As
shown in Table 1 (reproduced from TBSRTC), the system recognizes six
diagnostic categories and provides an estimated cancer risk for each, based on
identification of cancer in a subsequent nodule resection.

**TABLE 1**

**Bethesda Reporting System Categories for Thyroid Nodule Cytology and Risk of
Malignancy**

|

**Risk of malignancy if  
NIFTP = Cancer (%)**

|

**Risk of malignancy if  
NIFTP = Cancer (%)**  
  
---|---|---  
  
I. Nondiagnostic or Unsatisfactory

|

5-10%

|

5-10%  
  
II. Benign

|

0-3%

|

0-3%  
  
III. Atypia/Follicular Lesion of Undetermined Significance

|

6-18%

|



10-30%

  
  
IV. Follicular neoplasm or suspicious for a follicular neoplasm

|

10-40%

|

25-40%  
  
V. Suspicious for malignancy

|

45-60%

|

50-75%  
  
VI. Malignant

|

94-96%

|

97-99%  
  


A repeat aspiration with ultrasound guidance is recommended for nondiagnostic
Bethesda category I samples with excision considered for persistently non-
diagnostic or unsatisfactory nodules.23 Category II almost always results in
conservative surveillance as data continue to support a low false-negative
rate (<3%). Category VI, malignant, is used whenever features are conclusive
for malignancy with additional comments used to subclassify the malignancy. In
this case, near-total thyroidectomy or lobectomy is recommended. While this
system has been successful in distinguishing benign nodules from malignant,
approximately 20-25% of thyroid nodule FNA results are reported as
indeterminate or suspicious (Bethesda III-V) and nodules in these categories
carry a risk of malignancy from ~10-75%.23 In addition, intra- and inter-
observer variability has been reported leading to differences in
classification and the risk of malignancy (ROM) for these categories.24,25

Despite advances in technique, thyroid surgery is not without adverse effects
including general complications such as postoperative fever, infection,
hemorrhage, and cardiopulmonary events as well as thyroid-specific
complications including vocal cord/fold paralysis and hypoparathyroidism or
hypocalcemia. Thyroid-specific complications have been reported in more than
10% of patients and are significantly higher in patients greater than 65 years
of age.26 As such, there has been a concerted effort to develop technologies
to improve the classification and risk stratification of indeterminate and
suspicious thyroid nodules as part of the surgical decision making process.

**Molecular Testing**

Current clinical guidelines, including the ATA Management Guidelines for Adult
Patients with Thyroid Nodules and Differentiated Thyroid Cancer, and The
National Comprehensive Cancer Network (NCCN) Thyroid Carcinoma guidelines
endorse the use of molecular tests to further risk stratify patients with
indeterminate (Bethesda III and IV) thyroid nodule cytology results, as well
as their use in identifying cancer types with challenging cytology such as
MTC.16,27 Molecular tests can be broadly grouped into "rule-out" tests
designed to identify benign nodules thereby placing the patient on
surveillance and avoiding surgery, "rule-in" tests that aim to predict the
aggressiveness of malignancy and aid in surgical decision making and "general"
tests that can act as both rule-in and rule-out. Most currently offered tests
utilize Next Generation Sequencing (NGS) methodologies to either assess
characteristic gene expression profiles (GEP) or genomic sequence variant
profiles that are known to be associated with malignancy.

**First generation Tests**

The Afirma gene expression classifier (AGEC) is an early GEP test that was
developed as a rule-out.28 The landmark publication in 2012 described the test
validated against the gold standard histopathology of known benign or
malignant thyroid tissue and classified indeterminate thyroid nodules into
benign or suspicious using a proprietary algorithm based on gene expression
signatures.28 The algorithm assesses the expression of 142 primary genes plus
25 additional genes that filter out rare neoplasms such as medullary carcinoma
and renal carcinoma as the sample is processed through a series of
"cassettes." This prospective study examined 265 of 577 indeterminate nodules
from 4812 FNAs (5.5%) collected from 3789 patients at 43 clinical sites over a
19-month period. The AGEC correctly called 78 of 85 malignant samples
suspicious for a sensitivity of 92% and 93 of 180 benign samples were called
correctly for a specificity of 52%. These percentages were relatively
consistent regardless of the sample category. The prevalence of malignancy
(POM) was 24% and 25% for Bethesda category III and IV nodules respectively,
yielding a negative predictive value (NPV) of 95% and 94% respectively.
Because the POM for category V was much higher at 62%, the respective NPV was
85%. These data suggested that the AGEC could rule out malignancy in over 90%
of indeterminate category III and IV nodules. Since then, the test has
garnered wide acceptance in clinical practice and as described above, the
approach has been recommended by professional associations.16,27

Many follow up reports including multiple meta-analyses on the performance of
the AGEC have been published.29-31 Silaghi et al. summarized 25 studies
involving 4,538 indeterminate nodules of 4,424 patients who had been evaluated
using the AGEC test from May of 2009 to June of 2018.30 The overall
sensitivity and specificity across all studies was 97% and 19% respectively
with an NPV of 91% and positive predictive value (PPV) of 39%. However, most
of the reports are retrospective from single centers and demonstrate variable
test performance among institutions. Some reports indicate variability amongst
institutions that differ in POM of indeterminate nodules.32,33 For example, in
a study comparing the AGEC-benign call rate between Memorial Sloan Kettering
Cancer Center (MSK), a tertiary referral cancer center with a POM of 30-38%,
and Mount Sinai Beth Israel (MSBI) a comprehensive health system with a POM of
10-19%, Marti et al found that the NPV at MSK was 86-92% yet 95-98% at MSBI.
Conversely the PPVs of GEC-suspicious results were 57.1% and 13.3%
respectively with 86% (18/21) of resected GEC-suspicious nodules at MSBI being
ultimately benign on final pathology. This data matched closely to the
predicted PPVs and NPVs and highlights the importance of knowing the POM at
each institution.33 Valderrabano et al reported that the low resection rate of
GEC-benign nodules makes the false-negative and NPV impossible to calculate
and the only reliable metrics of benign call rate (BCR, the proportion of
nodules tested with a GEC-benign result) and PPV suggested that the initial
cohort study is not representative of the populations to which the AGEC was
subsequently applied.34

In 2011, Nikiforov et al. reported on the efficacy of a gene hot spot panel in
967 FNA samples from indeterminate nodules for variants that commonly occur in
thyroid cancer such as _BRAF_ p.V600E, _KRAS_ codons 12/13, _NRAS_ and _HRAS_
codon 61 and _RET_ and _PAX8_ fusions establishing that molecular profiling
using FNAs of thyroid nodules can aid in malignancy identification as a rule-
in test.35 This report was followed by further clinical validation studies on
Bethesda III and IV nodules using ThyroSeq v2 (TSv2), a panel consisting of
additional variant hotspots in genes known to be drivers in thyroid
carcinogenesis as well those that develop late with expression analysis of an
additional eight genes to determine cell type composition. The larger number
of variants examined resulted in a higher sensitivity than the original seven
gene panel as well as a higher NPV. In a study of 143 FNA samples from
patients with Bethesda category IV nodules with known surgical outcomes, the
TSv2 test demonstrated a sensitivity and specificity of 90% and 93%
respectively with a PPV of 83% and an NPV of 96%.36 Similar results were
obtained for category III nodules.37 However, like the AGEC, variability
across multiple institutions has been reported.38-40 For example, in a
retrospective analysis of 273 category III and IV nodules from four different
institutions, Marcadis et al. reported variation in test performance and
diagnoses.38 Although sensitivity was similar to what was originally reported
by Nikiforov, the specificity was lower (52% vs. 93%). This led to a range of
PPVs from 22%-43% across the institutions which is lower than what was
originally reported at 83%. A PPV of 22% was reported by Taye et al. with a
PPV of 9% (2/22) and 7% (1/15) across all _RAS_ and _NRAS_ mutations
respectively. The authors noted that many genetic alterations, such as those
in the _RAS_ family, appeared to be nonspecific for malignancy and positive
reports should be interpreted with care.39

**Second generation tests**

Updated versions of both rule-in and rule-out test types have been developed.
The AGEC was replaced by the Afirma Genomic Sequencing Classifier (AGSC) which
analyzes the entire RNA transcriptome rather than a subset of genes and
includes classifiers to identify samples with _BRAF_ or _RET_ alterations, as
well as characteristic MTC, PTC and HTC profiles.41 These tests use a
proprietary algorithm to classify an indeterminate nodule as benign or
suspicious. Thyroseq v3 (TSv3) is an expanded version of TSv2 containing
variant targets in 112 genes as well as copy number alterations (CNAs) in
multiple genomic regions and expression analysis of 19 genes. Results are
reported as positive (high probability of cancer/NIFTP) or negative (low
probability of cancer/NIFTP).42 Positive samples are further classified into
high, intermediate and low molecular risk groups based on the variant(s)
identified.

Multiple reviews have been performed on these second-generation tests and
describe increased performance over their predecessors.30,43,44 In Lee et al.,
preliminary pooled studies demonstrated that both assays, AGSC and TSv3, have
a high sensitivity (96% and 95% respectively) and high NPV (96% and 92%
respectively) demonstrating that either test type can be used to rule out
malignancy.43 The AGSC and TSv3 were reported to have a specificity of 53% and
50% with a PPV of 63% and 70% respectively. Although this represents an
increase in specificity for the AGSC (12% to 53%) the specificity for TSv3
compared to TSv2 went down (78% to 49.6%). However, the specificity of the
tests ranged considerably across multiple studies particularly from single
centers suggesting inter-institution variation similar to what was seen in the
first-generation tests. Silaghi reported similar results.30 Livhits et al
performed a randomized clinical trial across nine sites by using both the AGSC
and the TSv3 in practice on a rotating monthly basis.45 Of the 346 samples
ultimately tested, 189 and 157 were randomized to the AGSC and TSv3
respectively. For the AGSC test, 19 nodule samples were insufficient for
testing, 107 (53.2%) were classified as benign and 73 (36.3%) as suspicious.
Twelve of the benign samples were surgically resected and histopathologically
classified as benign. Fifty-eight of the suspicious samples were resected and
revealed NIFTP in 10 (17.2%) and malignancy in 21 (36.2%). The TSv3 test
identified 103 (60.2%) negative nodules, 60 (35.1%) positive, and seven
insufficient for testing. Eleven negative nodules were resected, and one was
found to be a minimally invasive Hurthle cell carcinoma with capsular invasion
only that was resected due to growth during the surveillance period. Of the
positive nodules, 49 (81.7%) were resected and histopathological results
revealed NIFTP in 11 (22.4%) and malignancy in 20 (40.8%). These data
demonstrated high sensitivity (97-100%) and reasonably high specificity
(80-85%) for both tests and diagnostic surgery was avoided in approximately
half of the patients in the study.45 However, consistent with similar studies,
nodules with benign/negative results were assumed to be benign in the absence
of histopathological confirmation. Therefore, to further assess the false
negative rates of the AGSC and TSv3, Kim et al. performed a prospective study
of a single center in patients surveilled over a median of 34 months (range
12-60).46 They reported that of the 217 indeterminate nodules initially
reported with negative or benign results 14 (8%) underwent immediate resection
and were all confirmed to be benign. Of the 147 that remained on continued
surveillance, 15 were resected during the surveillance period. The minimally
invasive Hurthle cell carcinoma initially found to be negative by TSv3,
remained the only false positive. Of the 133 test positive nodules, 97
underwent immediate resection and 59 were determined to be cancerous and of
those that were initially surveilled, 16 ultimately underwent delayed surgery
with an additional nine found to be malignant. These data reaffirm the high
sensitivity rate previously reported for both assays.46

**Molecular profiles**

The variants identified in a nodule can also predict the risk and/or class of
malignancy. For example, nodules with "driver" mutations such as _BRAF_
p.V600E or pathogenic variants in _RET_ have a higher probability of
malignancy than those carrying _RAS_ or _RAS_ -like variants.16 Tumors
harboring _BRAF_ p.V600E are generally classic PTC that frequently involve
regional lymph nodes with a higher rate of metastasis, and _RET_ mutations are
present in all inherited MTCs and 6-10% of apparent sporadic disease.15,47-50
In contrast, _RAS_ alterations ( _KRAS/NRAS/HRAS_ ) are the most frequently
identified in indeterminate thyroid nodules. However, unlike _BRAF_ p.V600E,
the utility of detecting _RAS_ alterations remains uncertain. In a systemic
review of 35 studies examining _RAS_ mutations published between 2000 and
2015, Najafian et al. reported a prevalence of _RAS_ mutations in 0-48% of
benign nodules and 10-93% of malignant nodules across the studies.51 In a
study of over 1500 patients, Yip et al. reported an indolent clinical course
and nearly 100% disease free survival at five years for patients with _RAS_
-positive nodules.50 Guan et al. also reported that although _RAS_ variants
were the most frequent alterations detected in more than 500 fine needle
biopsies, they provided poor value for prediction of TC since most _RAS_
alterations presented in benign nodules and NIFTPs (59% and 13%
respectively).52 However, the presence of a "second-hit" in another gene such
as _TERT_ or _TP53_ significantly increased the risk of malignancy.

Key decisions that must be made in the care of patients with suspicious
thyroid nodules include whether to surgically intervene, the extent of surgery
(such as a full vs. partial thyroidectomy), and the selection of therapeutic
intervention. Tests designed to rule-out a disease should have high
sensitivity and NPV, and those designed to rule-in should have high
specificity and PPV with tests that provide both high rule-in and rule-out
probabilities being ideal. A high sensitivity and NPV is necessary for rule-
out tests on indeterminate nodules that would otherwise be surgically removed.
However, a test's efficacy as a rule-out is based on its NPV which changes
based on institution and POM. Rule-in tests with high specificity should only
be considered if the results will alter the management of the patient.
Knowledge of the disease prevalence in indeterminate thyroid nodules at any
institution is critical for clinicians to anticipate the diagnostic
performance of a test.

NCCN guidelines state that molecular testing may be useful to allow
reclassification of Bethesda III and IV nodules.27 In addition, NCCN
acknowledges the challenges of diagnosing MTC and molecular tests may assist
in identification as well as provide information to aid in extent of surgery
such as a lobectomy vs. total thyroidectomy. However, the guidelines also note
that the results of the tests should be interpreted in the context of
clinical, radiographic and cytologic features of each patient. The most recent
ATA guidelines were published in 2016 and state that molecular testing may be
useful after consideration of clinical and sonographic features if such data
is expected to alter surgical decision making.16 They also recommend that if
molecular testing is being considered, either rule-out or rule-in, patients
should be appropriately counseled regarding the benefits and limitations of
these tests and note that the utility of these types of tests is strongly
influenced by the prevalence of cancer in the population being tested. The
American Association of Endocrine Surgeons (AAES) note that molecular testing
is primarily utilized as a preoperative adjunct to refine the cancer risk in
cytologically indeterminate thyroid nodules, and that molecular testing is
acceptable for Bethesda III and IV nodules. However, if thyroidectomy is
preferred for clinical reasons, gene expression profiles are unnecessary.
Instead, comprehensive DNA tumor profiling that can guide treatment should be
performed on the resected tissue. The AAES also points out the importance of
knowledge of institutional malignancy rates.53

Reports on second generation molecular tests for assessing indeterminate
thyroid nodules in general show improved performance with increased
sensitivity and specificity over their predecessors and may be considered
"general" tests with the ability to both rule-out the need for surgery if
negative and rule-in the extent of treatment if positive. However, the
prevalence of variants in _RAS_ genes in benign nodules suggests the need for
caution in decision making. Relying on the presence of a single _RAS_ mutation
to drive clinical decision making may result in unnecessary treatment.
Additional factors such as biological behavior of the nodule, secondary
variants and other clinical indications should be considered in nodules
positive for _RAS_ variants.

Given the cost and risks associated with thyroid surgery, the clinical
decision making that goes into the extent of surgery, additional follow up
management, and published guidelines, this contractor finds that molecular
tests that aid in medical decision making for thyroid nodules of Bethesda
Categories III-V are reasonable and necessary. Specifically, this contractor
recognizes improved outcomes by safely precluding unnecessary surgical
procedures with these services as well as better selecting patients who may
benefit from such procedures. Reference to specific tests in this document
does not imply coverage by this contractor.