**Indications and Limitations of Coverage**

****

The use of molecular testing (MT)* to assess thyroid nodules may be considered
medically necessary **ONCE** per nodule workup when **ALL** of the following
criteria are met:

  1. Thyroid fine-needle aspiration (s) (FNA) performed secondary to established indications based on ultrasound characteristics, size and clinical findings (1)
  2. Presence of indeterminate thyroid FNA cytopathology described as (1,2)
    1. Atypia of undetermined significance (AUS) or follicular lesion of undetermined significance (FLUS) (i.e., Bethesda category III), or
    2. Follicular neoplasm (FN) or suspicious for a follicular neoplasm (SFN) (i.e., Bethesda category IV)
  3. The need for thyroidectomy is unclear after consideration of clinical, imaging, and cytologic features (1,2)
  4. Local institutional malignancy rates are known and used for optimal extrapolation of results to thyroid cancer risk (1)
  5. Informed patient willing to potentially undertake surveillance (1)

*The scope of this LCD is limited to tests submitted on claims to NGS jurisdictions J6 and JK.

Thyroid cancer is the most common endocrine malignancy, with over fifty
thousand new United States diagnoses in 2020 (3). Thyroid nodules are
exceedingly common with prevalence rates of up to 68%, with higher frequencies
in the elderly (4). While the majority (85-93%) of thyroid nodules are benign,
diagnostic testing (history and physical, laryngoscopy, hormone and chemistry
analysis, ultrasound, CT, FNA, and surgical excision) is required to confirm.
Over 600,000 thyroid FNAs are performed every year in the United States, and
the number has been increasing annually by 16% (5). Despite this diagnostic
gauntlet, approximately 20% of FNA results are classified indeterminate
(Bethesda III/IV) with a 10-40% risk of malignancy (6). Cancer rates vary
widely by institution, ranging from 6-48% for Bethesda III and 14-34% for
Bethesda IV. Repeat FNA of Bethesda III nodules should be strongly considered
as it leads to a more definitive reclassification in 60-65% (1). Previously,
the majority of patients with an indeterminate FNA, confirmed on repeat
aspiration, had diagnostic thyroid surgery (usually lobectomy), with most
(75-95%) ultimately confirmed to be benign (6). Rates of thyroid surgery-
specific postoperative complications (recurrent laryngeal nerve injury,
permanent hypoparathyroidism, and postoperative hematoma) in high-volume
institution studies range between 0.4-7.4%, but a population-based study found
it as high as 12.3% (7).

In 2012, molecular marker testing (MT) became widely available as a potential
method to augment risk stratification of indeterminate FNA results, ideally
reducing the need for diagnostic thyroid surgery or completion thyroidectomy,
with their attendant risks and costs. A patient with a low MT malignancy risk
is potentially recommended for surveillance with serial ultrasounds to ensure
nodule stability. Conversely, a high malignancy MT result could strengthen a
recommendation to move forward with surgical removal (lobectomy or total
thyroidectomy). Molecular profiling includes genomic alterations (such as
point mutations, insertions, and deletions), gene fusions resulting in
rearrangements or translocations, copy number variations, RNA-based gene
expression, and/or micro-RNA (miRNA) expression (1).

In 2016, the nomenclature of encapsulated follicular variant of papillary
thyroid cancer was changed to noninvasive follicular thyroid neoplasm with
papillary-like nuclear features (NIFTP) in recognition of its highly indolent
nature. Thus, the value of MT may arise both from the avoidance of surgery and
the fact that surveillance is now a safer and more informed option (8).
Conversely, given clinical guideline recognition that more limited cancer
operation can lead to equivalent outcomes, the impact of molecular testing in
directing the extent of surgical resection is diminished (9).



_Thyroseqv3 (CBLPath, Inc.; Rye Brook, NY)_

Thyroseqv3 is a targeted next-generation sequencing test that interrogates
selected regions of 112 thyroid cancer-related genes for point mutations,
insertions/deletions, gene fusions, copy number alterations, or gene
expression alterations (5). A "genomic classifier" assigns a value to each
detected genetic alteration based on the strength of association with
malignancy: 0 (no association with cancer), 1 (low cancer probability), or 2
(high cancer probability). A score calculated for each sample is a sum of
individual values of all detected alterations, with scores 0 and 1 accepted as
test negative (score 1 commercially reported as currently negative) and scores
2 and above as positive.

This evidence summary is limited to the current version of Thyroseq (version
3). Also excluded are publications combining results from multiple Thyroseq
versions in which version 3 results could not be specifically isolated
(8,10,11).

In a multicenter prospective, blinded, clinical validation study of
ThyroSeqv3, including 247 Bethesda III and Bethesda IV nodules in which both
pathologist and clinicians were blinded to MT results, sensitivity was 94%
(95% CI, 86%-98%) and specificity 82% (95% CI, 75%-87%). With a cancer/NIFTP
prevalence of 28%, the negative predictive value (NPV) was 97% (95% CI,
93%-99%), the positive predictive value (PPV) was 66% (95% CI, 56%-75%), with
a benign call rate (BCR) of 61% (5). The observed 3% false-negative rate was
similar to that of benign cytology, and the missed cancers were all low-risk
tumors. Results of 10 Bethesda V nodules are not separately reported.

In another Thyroseqv3 validation study, 238 surgically removed tissue samples
were used as a training set and 175 indeterminate (Bethesda III, n=84;
Bethesda IV, n=74; Bethesda V, n=17) FNAs were used as a validation set (12).
The training set sensitivity was 93.9% (95% CI, 88.4%-96.9%), the specificity
89.4% (95% CI, 81.1%-94.3%), with an accuracy of 92.1% (95% CI, 87.8%-95.0%).
The validation set sensitivity was 98.0% (95% CI, 92.9%-99.4%), the
specificity 81.8% (95% CI, 71.8%-88.9%), with an accuracy of 90.9% (95% CI,
85.7%-94.3%). The sensitivity and specificity for Hurthle cell lesions in the
training set was 92.9% (95% CI, 80.52%-98.50%) and 69.3% (95% CI,
48.21%-85.67%), respectively. A separate case study also showed a benefit in
indeterminate Hurthle cell cytopathology (13).

In an independent, single-center, non-blinded observational study, a total of
50 Bethesda III/IV cytologically indeterminate nodules underwent ThyroSeqv3
testing (14). Molecular analysis yielded 20 (40%) "positive" results and 24
(48%) "negative" results. Six (12%) results were classified as "currently
negative" or "negative but limited." All 20 "positive" patients underwent
surgery, as well as both "currently negative" patients (n=2) and one patient
with a "negative but limited" result (n=1). All 26 "negative" patients and one
patient with a "negative but limited" result (n=1) continued with
surveillance. In total, 23 (46%) patients underwent surgery and 27 (54%)
patients were followed with conservative management. BCR was calculated as
("negative" and "currently negative")/total, resulting in a BCR of 58%.
Ninety-one percent (20 of 22) of the resected target nodules were malignant on
final pathology. Since surgery was not performed on test-negative patients,
test specificity, sensitivity, and NPV were not available.

One retrospective 2019 review of 224 thyroid nodules with available ThyroSeqv3
Bethesda III or IV cytology had a BCR rate of 75% (15). In a hypothetical
cost-effectiveness analysis, ThyroSeqv3 was superior to diagnostic lobectomy
for indeterminate (Bethesda III/IV) nodules (16).



Guidelines emphasize reserving MT for thyroid nodules with equivocal clinical,
cytopathologic, and radiographic factors. The 2015 American Thyroid
Association (ATA) guideline for management of thyroid nodules has several
cautious recommendations specific to MT (4): 1/ If molecular testing is being
considered, patients should be counseled regarding the potential benefits and
limitations of testing and about the possible uncertainties in the therapeutic
and long-term clinical implications of results. (Strong recommendation, Low-
quality evidence); 2/ For nodules with AUS/FLUS cytology, after consideration
of worrisome clinical and sonographic features, investigations such as repeat
FNA or molecular testing may be used to supplement malignancy risk assessment
in lieu of proceeding directly with a strategy of either surveillance or
diagnostic surgery (Weak recommendation, Moderate-quality evidence); 3/
Diagnostic surgical excision is the long-established standard of care for the
management of FN/SFN cytology nodules. However, after consideration of
clinical and sonographic features, molecular testing may be used to supplement
malignancy risk assessment data in lieu of proceeding directly with surgery
(Weak recommendation, Moderate-quality evidence); and 4/ When surgery is
considered for patients with a solitary, cytologically indeterminate nodule,
thyroid lobectomy is the recommended initial surgical approach. This approach
may be modified based on clinical or sonographic characteristics, patient
preference, and/or molecular testing when performed. (Strong recommendation,
Moderate-quality evidence).

More recent guidelines are even more circumspect due to a combination of
interim factors (9). The American Association of Endocrine Surgeons (AAES)
2020 guidelines echo recent heightened concerns with MT (1). They cite the
following qualifiers: 1/ "follow-up independent studies have often reported
diminished utility;" 2/ "providers and patients may also find it challenging
to interpret MT results...potentially leading to over- or under-treatment;" 3/
"patient willingness to continue surveillance," must be considered before
obtaining MT; 4/ use of MT results to make clinical decisions relies on PPV
and NPV which are contingent on regional and institutional cancer prevalence
for each cytology category; and 5/ "because NIFTP decreases the risk of true
malignancy for the indeterminate Bethesda categories the PPV of all MT will be
impacted." In other words as to the last point, given that molecular tests
were developed and validated prior to this re-designation (and thus designed
to classify this potential benign pathology as malignant), their performance
measures have been shown to deteriorate significantly when the NIFTP
designation is incorporated in the classification of indeterminate nodules
(9). The impact of NIFTP reclassification is not trivial, as its average
prevalence within indeterminate thyroid nodules is estimated to be 61% (range,
33-88%) (17,18).

AAES guidelines cite three specific MT recommendations: 1/ If thyroidectomy is
preferred for clinical reasons, then MT is unnecessary. (Strong
recommendation, moderate-quality evidence); 2/ When the need for thyroidectomy
is unclear after consideration of clinical, imaging, and cytologic features,
MT may be considered as a diagnostic adjunct for cytologically indeterminate
nodules. (Strong recommendation, moderate-quality evidence); and 3/ Accuracy
of MT relies on institutional malignancy rates and should be locally examined
for optimal extrapolation of results to thyroid cancer risk. (Strong
recommendation, moderate-quality evidence). Use of MT for Bethesda V nodules
is not endorsed as they cite validation and utility studies as lacking.
Specifically with respect for MT to guide extent of surgery, they note:
"Further study will determine if genotype provides information that has not
already been obtained clinically, by US imaging, and/or by cytologic
classification, as well as determine if altering the initial extent of surgery
based on MT results will affect outcomes." UpToDate also restricts potential
MT use to Bethesda III and IV nodules (6).

National Comprehensive Cancer Network (NCCN) guidelines have similar criteria
(2). They make a point of adding that: "Molecular diagnostics may be useful to
allow reclassification of follicular lesions (i.e., follicular neoplasm, AUS,
FLUS) as either more or less likely to be benign or malignant based on the
genetic profile....," but "should be interpreted with caution and in the context
of clinical, radiographic, and cytologic features of each individual patient."

In summary, NGS supports judicious use of molecular testing of thyroid nodules
in alignment with current guidelines. We look forward to additional
independent, prospective studies that better define clinical utility,
especially in the context of recent improvements in imaging (Thyroid Imaging,
Reporting, and Data System (TIRADS) algorithm) and cytologic classification.