National Government Services (NGS) will not provide coverage for the oncology
(gastrointestinal neuroendocrine tumors) real-time PCR expression analysis of
51 genes, utilizing whole peripheral blood, algorithm reported as a nomogram
of tumor disease index for its use in treating neuroendocrine tumors. It has
not been accepted by most neuroendocrine treatment guidelines. Most important,
this test has not been shown to result in improved outcomes for Medicare
beneficiaries and thus is not medically necessary.

Multianalyte Assays with Algorithmic Analyses (MAAA) codes are Administrative
codes. Thus, they do not require adherence to Category I Code Criteria or AMA
review for clinical utility.  Such codes are unique to a single clinical
laboratory or manufacturer.  These codes are based on various assays (DNA,
RNA, immunoassay, or other biomarkers) and may include other elements such as
age and sex. An algorithm (often proprietary) is then used on the assay data
to assign a single, patient-specific value that indicates diagnosis,
mitigation, treatment, or prevention of disease.  Such a code is requested by
the laboratory or manufacturer performing the test.  Unlike Category I CPT
codes, Category II CPT codes, or Category III CPT codes, these are found only
in the Appendix O of the _CPT Codebook_ (1).  It may or may not be FDA
approved.  Per CPT 2018, these codes "are provided as an administrative coding
set to facilitate accurate reporting of MAAA services.  The minimum standard
for inclusion in this list is that an analysis is generally available for
patient care.  The AMA has not reviewed procedures in the administrative
coding set for clinical utility" (1).

Neuroendocrine tumors are a heterogeneous group of neoplasms (2) that can
occur in the gastrointestinal system (including the pancreas), the lungs, and
other organs.  Previously called carcinoid tumors, these are most commonly
referred today as neuroendocrine tumors (NET) or neuroendocrine neoplasms
(NEN).  Despite new promising effective therapies that have evolved in the
past few years for the well-differentiated gastroenteropancreatic
neuroendocrine tumors (GEP-NETs), advanced metastatic gastroenteropancreatic
neuroendocrine tumors continue to be an incurable disease with poor prognosis.
There is no standard guideline regarding the optimal selection and sequencing
of treatment strategies for patients with advanced GEP-NETs (2).

"Traditionally, neuroendocrine tumors are described as arising from the
foregut (bronchopulmonary, thymus, gastric, proximal duodenum, pancreas),
midgut (distal duodenum, jejunum, ileum, ascending colon), or hindgut (distal
colon, rectum). Primary tumors arising from these different anatomic zones
differ in their tumor secretions. For example, tumors originating in the
midgut secrete serotonin and are more likely than tumors of other origins to
present with carcinoid syndrome. Carcinoid syndrome, when present, may include
the following symptoms: flushing (94%), diarrhea (78%), abdominal cramping
(50%), valvular heart disease (50%), telangiectasia (25%), wheezing (15%), or
edema (19%).  Pulmonary tumors are less likely to secrete serotonin (although
they may secrete the serotonin precursor, 5-hydroxytryptophan) and more likely
to secrete histamine. Although foregut tumors can present with flushing and
wheezing, the full carcinoid syndrome with diarrhea is unusual. Hindgut tumors
rarely secrete serotonin or cause carcinoid syndrome.  In addition, the
clinical presentation may vary between tumors of the same originating site,
based on the tumor grade and stage. Some biochemical tests have been suggested
as markers of tumor grade or differentiation, for example, neuron-specific
enolase. Although only about 10% of NET present with features of carcinoid
syndrome, this number increases with greater tumor bulk (later stage),
especially when liver metastatic disease increases" (3).

In contrast to gastroenteropancreatic neuroendocrine tumors (GEP-NETs), lung
carcinoids (LCs) and large cell neuroendocrine lung carcinomas (LCNELC) are
less frequent. Lung carcinoids are well-differentiated neuroendocrine tumors
and account for 8% of lung neuroendocrine tumors and for 1-2% of all lung
tumors (3). They are divided into the low-grade typical carcinoids with
minimal mitotic activity (<2 mitoses per 2-mm2 field) without any necrosis,
and the intermediate-grade ACs with more mitoses (2-10 per 2-mm2 field) and
presence of focal necrosis. On the other hand, large cell neuroendocrine lung
carcinomas are poorly differentiated lung neuroendocrine tumors or
neuroendocrine carcinomas with morphological appearance resembling non-small
cell lung cancer (NSCLC) but biological similarities with small cell lung
cancer (SCLC). Large cell neuroendocrine lung carcinomas account for
approximately 12% of lung neuroendocrine tumors and are found in 2.1-3.5% of
surgically resected bronchopulmonary neoplasms. Another critical aspect of
lung carcinoids that affects treatment decisions is functionality, which means
their capability of causing secretory syndromes that are named paraneoplastic
when the secretory component is not derived from the expected tissue of
origin. However, the majority are non-secretory and usually present similar to
other lung cancers (cough, hemoptysis, fever). Paraneoplastic syndromes
develop in approximately 10-15% of cases, the most common being carcinoid
syndrome or Cushing's syndrome secondary to ectopic serotonin or
adrenocorticotropic hormone (ACTH) hypersecretion by tumor cells (4).

The main determinants of treatment planning are the originating site of the
tumor, histologic subtype of the tumors and extent of the disease.
Historically carcinoid tumors (or neuroendocrine tumors [NET] or
neuroendocrine neoplasms [NEN]) have been called rare tumors, but through
registries, it is apparent that this is no longer correct. Their annual age-
adjusted incidence increased from 1.09 per 100,000 person in 1973 to 6.98 per
100,000 persons by 2012. Neuroendocrine tumors are classified histologically
based on tumor differentiation (well or poorly differentiated) and tumor grade
(grades 1-3). Most neuroendocrine tumors fall into 3 broad histological
categories: well-differentiated, low-grade (G1); well-differentiated,
intermediate-grade (G2); and poorly differentiated, high grade (G3)(5).
Survival depends on many factors including the site of the tumor, whether the
tumor is localized, and the grade of the tumor (6).

Chromogranin A is a secreted protein that may be elevated in patients with
neuroendocrine tumors; elevated levels have been associated with poorer
prognosis. Currently, Chromogranin A (CgA) is a well-established marker for
diagnosis and follow up of patients with gastroenteropancreatic neuroendocrine
neoplasms (GEP-NEN). Recently, it has been shown that plasma levels of CgA
correlate with tumor load and predict survival of patients with neuroendocrine
neoplasms (NEN) of the small bowel but CgA plasma concentration does not
appear to mirror tumor burden or prognosis in patients with neuroendocrine
tumors of the colon and rectum (7). While chromogranin A testing is recognized
by the National Comprehensive Cancer Network (NCCN )(5) Neuroendocrine and
Adrenal Tumors, "oncology (gastrointestinal neuroendocrine tumors) real-time
PCR expression analysis of 51 genes, utilizing whole peripheral blood,
algorithm reported as a nomogram of tumor disease index" (such as NETest) is
not listed by National Comprehensive Cancer Network Guidelines, although
"Neuroendocrine Tumor Biomarkers: Current Status and Perspectives" by Dr.
Modlin, Oberg, Taylor, Drozdov, Bodei, and Kidd is listed as a reviewed
reference (8). NCCN notes, "More research is required, however, before these
and other new molecular assays are routinely used in the clinic. A
multinational consensus meeting of experts concluded that, to date, no single
currently available biomarker is sufficient as a diagnostic, prognostic, or
predictive marker in patients with neuroendocrine tumors" (5).

In addition, the guidelines note, "The molecular basis of neuroendocrine
tumors remains poorly understood, and additional molecular predictors of
outcome remain investigational. A recent study found that overexpression of
mammalian target of rapamycin (mTOR) or its downstream targets was associated
with shorter overall survival in 195 neuroendocrine tissue samples (15% were
located in the pancreas; 85% were GI carcinoids). Small bowel carcinoid tumors
have been found to have recurrent mutations in the cyclin-dependent kinase
inhibitor, CDKN1B(p27) and loss of CDKN1B expression has been reported to be
an adverse prognostic factor in gastroenteropancreatic neuroendocrine tumors.
Circulating tumor cells (TCs) have also been studied as possible prognostic
markers, based on the idea that tumor cells in the blood would be indicative
of more disseminated disease. A recent study found that the presence of
greater than or equal to 1 CTC in 7.5 mL of blood was independently associated
with worse progression-free survival (PFS) and overall survival in patients
with varying pre-treated metastatic neuroendocrine tumors from various primary
sites.

Related, Crabtree in her recent review article noted, "Gastrointestinal
neuroendocrine tumors are a very heterogeneous group of tumors. Targeted
therapies are available to treat these tumors but despite the many clinical
approaches to neuroendocrine tumors, and beyond surgery for localized disease,
there is little consensus on first line therapy. Results of ongoing clinical
trials will better inform patient management with respect to selection,
timing, duration, and combination of available therapies, and immunotherapy
holds great promise for NETs and other cancers" (9).

Other prognostic blood-based biomarkers for neuroendocrine tumors have been
proposed. MiRNAs are short (approximately 22 nucleotides) RNA sequences that
have been shown to broadly regulate gene expression at a post-transcriptional
level, by binding to the 3' region of target RNAs, resulting in mRNA
degradation and inhibition of translation. A recent small study suggested that
elevated circulating levels of two such short RNA sequences and low levels of
another are characteristic in patients with metastatic small intestine
neuroendocrine tumors, and further suggests that levels of these miRNAs are
associated with overall survival (10).

Bodei et al. (11) PRRT Genomic Signature in Blood for Prediction of 177Lu-
octreotate Efficacy. This was submitted in a recent reconsideration request
and is being reviewed as a new reference. Neuroendocrine tumors (NETs) can be
treated with targeted therapies including somatostatin analogues everolimus,
sunitinib and targeted radionuclide therapies. Peptide receptor radionuclide
therapy (PRRT) utilized octreotide derivatives 177Lu-DOTA-Tyr3,
Thr8octreotide, or 177Lu-octreotate. This article discusses the prediction of
these therapies using the PRRT Predictive Quotient (PPQ). The results of the
PPQ are PRRT-responder or negative: PRRT-non-responder. Originally there were
178 patients enrolled but 20 were excluded. Median progressive free survival
(mPFS). Seventy-two were retrospective, 86 were in two different cohorts. The
overall accuracy was 94% with 95% being responders and 91% non-responders.
There was a difference in the tumor grade between the cohorts. There was an
evaluation of retrospective samples from the comparator cohorts. While there
was a significant difference in the accuracy of the testing between responders
and non-responders, this does not mean that this testing would necessarily
influence the treatment of patients, since many patients and physicians would
still use a PRRT (peptide receptor radionuclide therapy) despite the negative
testing. Most important, while median progressive free survival was different,
there was no evidence of true improved outcomes. Thus there is limited support
from this document for NETest. Of further note, the second author (Mark Kidd)
is an employee of Wren Laboratories and Dr. Modlin is a paid consultant for
Wren Laboratories.

Oberg et al. (12) Consensus on Biomarkers for Neuroendocrine Tumour Disease.
"The experts were of the opinion that such laboratory tests should be
initially undertaken at a single central laboratory." This consensus and this
recommendation for a single central laboratory was supported by Clifton Life
Sciences, which owns Wren Laboratories. In addition, Irvin M Modlin, Marianne
Pavel, David C Metz, Dik Kwekkeboom, Jonathan Strosberg, Timothy Meyer, Eric
Liu, and, James Goldenring (eight of the sixteen panelists) are listed as
having financial ties to Wren Laboratories. This has previously been submitted
and reviewed by this contractor in a prior reconsideration request. Thus there
is limited support from this document for NETest.

Kidd et al. (13) This is not a research article, but an explanation of NET
treatments. Of note, both authors are associated with Wren Laboratories. This
has previously been submitted and reviewed by this contractor in a prior
reconsideration request. This is not support for NETest.

Modlin et al. (14) Polymerase chain reaction (PCR) based 51-transcipt
signature test was evaluated in two groups of patients (125 prospectively
collected with known neuroendocrine tumors) and 29 patients with
gastrointestinal disease without known neuroendocrine tumors but taking oral
proton pump inhibitors and 50 controls. These were also tested with
chromogranin A (CgA). 123 (98.4%) of the known neuroendocrine tumor patients
had a positive test score while 50 (40%) had an elevated CgA. The cohort had
no positive test score but 24 had a positive CgA. This states that "More
pertinently, cancer center groups such as the National Comprehensive Cancer
Network (NCCN), have endorsed the use of high performance biomarkers
particularly in fields where there are no better alternative." This is a
misrepresentation of what was said and attributed to Reinke. Reinke had
actually noted "The NCCN acknowledges the harms of PSA testing; its
perspective is that PSA testing is the starting point for diagnosing prostate
cancer and that steps should be taken to improve its value. 'The Achilles'
heel of PSA testing is overdetection, and the USPSTF [United States Preventive
Services Task Force] got that ... 'But the USPSTF did not interpret the data
on PSA testing correctly.'" (15) In addition, the use of PSA testing has been
shown to give inappropriate information and is no longer recommended as a
screening test. This discussion of PSA is not related NETest. Furthermore, the
NCCN acknowledges chromogranin A as being the standard biomarker for
neuroendocrine tumors. A great concern is that the "Declaration of interest:
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported." However,
the paper states "This work was supported by Clifton Life Sciences" which owns
Wren Laboratories. Also Modlin I, Bodei L, Drozdov I, and Kidd M are listed as
being from Wren Laboratories. This has previously been submitted and reviewed
by this contractor in a prior reconsideration request. Thus there is limited
support from this document for NETest.

Perrier ND. (16) This was submitted in a recent reconsideration request to
this contractor and is being reviewed as a new reference. This paper is a
review of the history of MEN1 gene and its function and treatment. The NETest
is mentioned under "Prediction of response to therapy: Determination of the
effect of therapy is importantly needed. The NETest shows the potential value
of multidimensional tumor marker studies. Developed by Modlin and his team,
NETest is a 2-step multianalyte PCR-based circulating gene test of the mRNA
expression levels of 51 target genes. The test involves RNA isolation with
cDNA production. Measurement of these gene transcripts in blood can confirm
reduction in tumor burden, and quantification of the molecular signature over
time may provide information about the efficacy of treatment and amplify the
identification of nonresponders. The NETest is now marketed to manage gene
transcripts in blood." This review concludes with "Further pursuit of
prevention, prediction, pausing progression, with adjunct modalities for
diagnosis and roved options for systemic therapy will be beneficial. The time
for progress is now." This is not a robust support of the NETest, but is the
only paper with any support of NETest that does not appear to be written by
Dr. Modlin and/or Wren Laboratories. There is no mention of conflict of
interest: this subject is not brought up.

Singh et al. (17) Singh and his colleagues reported on the largest known
series examining outcomes of patients with fully resected
gastroenteropancreatic neuroendocrine tumors (GEP-NETs). As with other
researchers, he notes gastroenteropancreatic neuroendocrine tumors (GEP-NETs)
are widely heterogeneous malignant abnormalities. He notes that the natural
history of these tumors is poorly described, with little understanding of
recurrence patterns. Surveillance for resected GEP-NETs may include clinical
review, laboratory tests, and numerous medical and nuclear imaging modalities.
These modalities can increase patient anxiety, may be associated with
potential harm (eg, exposure to ionizing radiation), and have not been shown
to improve outcomes. Current guidelines vary widely in recommendations,
reflecting the lack of data. Information on the natural history and recurrence
of the disease may improve patient-centered follow-up of this population. He
hypothesized that GEP-NETs may recur over a longer time course compared with
other gastrointestinal malignant abnormalities. No conflict of interest
disclosures were reported. This does not support NETest.

Singh et al. (18) Cancer Treatment Reviews is an over-view of gastrointestinal
neuroendocrine tumors in Canada, using evidence based consensus. While three
of Dr. Modlin's older papers are cited concerning anatomic sites and secretory
syndromes, there is no mention of any markers, including NETest. There are a
number of authors who disclosed potential conflict of interests, although not
all had any. This paper does not support NETest.

Reichelmann et al. (19) This is a detailed review paper that discusses all
phases of the diagnosis, imaging, and treatment of neuroendocrine tumors. It
states, "Chromogranin A is acceptable, despite its limitations, as a general
marker of NETs. The sensitivity and specificity limitations of chromogranin A
may depend on the test used and clinical situations, such as secondary
hypergastrinemia, gastrinoma, atrophic gastritis, Helicobacter pylori
infection, use of proton pump inhibitors and liver or kidney dysfunction.
Elevated levels of chromogranin A may also be caused by other neoplasias,
diminishing its specificity."

"Recommendations

  * Chromogranin A can be used as a prognostic test [IIIC], and can be used for monitoring purposes [7] [IVC]. It should not be used alone to guide management.
  * Repeated chromogranin A measurements can be made to assess tumour response [8], although the results should not be used to determine management [IIIC]." 

Conflict of interest is not mentioned. NETest is not mentioned in this
article. No articles by either Dr. Kidd or Dr. Modlin are mentioned in the
references. This article does not support NETest.

Raphael et al. (20) This review article summarizes the classification,
presentation, diagnostic workup and treatment of neuroendocrine tumors. It is
interesting in that it is a general overlook of neuroendocrine tumors with the
emphasis on "helping generalists to facilitate timely diagnosis and
referrals." Thus while it gives an excellent overview and understanding of
neuroendocrine tumors, it does not get bogged down in pure research results,
and concentrates on understanding the pathology, symptoms, diagnosing and
treatments. For patients presenting with symptoms of functioning
neuroendocrine tumors (atypical carcinoid, carcinoid, Whipple triad,
Zollinger-Ellison, 4D syndrome, somatostatinoma, Vern-Morrison [WDHA
syndrome], and Cushing syndrome), biochemical testing should be targeted to
the specific syndrome. The usual classical testing is recommended. For those
without symptoms of nonfunctioning neuroendocrine tumors, "Chromogranin A is
the diagnostic biomarker of choice for neuroendocrine tumors. It has a high
sensitivity (53%-91%) but low specificity (<50%). The most common reasons for
false elevations include proton-pump inhibitors, renal insufficiency,
adenocarcinomas and severe arterial hypertension." The author concludes with
an Unanswered Question paragraph. "Several unanswered questions remain. What
are the best predictive and prognostic markers for patients with
neuroendocrine tumours? What is the optimal follow-up strategy for patients
with neuroendocrine tumours? How should the multiple new therapeutic options
be sequenced and/or combined? How do the new therapeutic options affect
patient quality of life and survivorship?" While one of Dr. Modlin's
publications is referenced, there is no mention of NETest. Dr. Raphael does
not disclose any "competing interests." Co-authors Law, Singh, and Chan have
several industrial ties. This paper does not support NETest.

Kidd et al. (21) In this study, the "goal was to advance from a linear
interpretation of disease activity based upon quantification of gene
expression (0-8 score) to an amplified algorithm that incorporated gene
clusters (GC) representative of NET neoplasia." The researchers had eight
samples of normal tissue, two being from small intestines, four being from
colon, and two being from rectums. These were the controls. There were 22
tumor samples (one stomach; four pancreas; ten small intestine; one appendix
and six rectum. Two of the pancreas tumors, five of the small bowel, and three
of the rectum were metastatic tumors; the others were primary tumors. Fifty-
one genes as per previous related works were analyzed. Quantification of gene
expression in gene clusters was undertaken by summation of individual gene
expression. Individual genes clusters that were significantly different
between stable disease and progressive disease and were elevated in
gastroenteropancreatic neuroendocrine tumor blood compared to control blood,
were included in an activity algorithm. Six gene clusters (SSTRome,
proliferome, metabolome, secretome (II), epigenome, and plurome) met this
criterion and were summated. The authors concluded that using this cluster
integrated algorithm, gene clusters differentiate stable disease from
progressive disease. They also concluded that gene expression in blood reflect
tissue gene expression and that "the NETest captured the biology of NET
neoplasia and that integrating these measurements of circulating gene
expression could accurately define clinical status." This study is from Wren
Laboratories. "Funding: This work was supported by Clifton Life Sciences."
"Declaration of interest: The authors declare that there is no conflict of
interest that could be perceived as prejudicing the impartiality of the
research reported." This is an interesting study that the later NETesting is
based upon. It does suffer from the same potential perception about conflict
of interest. It needs external validation. No evidence of improved outcomes
are noted. This has previously been submitted and reviewed by this contractor
in a prior reconsideration request. There is limited support for NETest.

Zandee et al. (22) Although this article is nine pages, it is a brief overview
of classification, presentation, diagnostic workup and treatment of
neuroendocrine tumors, but more in a historical prospective. Biomarkers are
not discussed, thus there is no mention of chromogranin A or NETest. There is
no mention of conflict of interest. This does not support NETest.

Sato et al. (23) This is another review paper concerning the increasing
incidence of gastric and duodenal neuroendocrine tumors, their clinical
features, their diagnosis, their histology, their treatment, and their follow-
up strategy. The paper specifically recommends chromogranin A levels including
in their follow up and using them for monitoring. There is no mention of
NETest, although several Modlin/Kidd papers are referenced. The authors
declared "no financial or other conflicts of interest." This article does not
support NETest.

Oberg et al. (24) This is not a true research paper. It is a consensus
statement by thirty-two researchers concerning NETest. This meeting's
"financial support was provided by Clifton Life Sciences." "All the authors
except Dr. Jensen and Dr. Krenning received reimbursement for accommodation
and traveling expenses to and from the NET Consensus Meeting as well as an
honorarium. Mark Kidd and Ignat Drozdov receive salary support from Wren
Laboratories. Ignat Drozdov did not attend the final meeting and was not
involved in the final voting. Mark Kidd did not vote on sections involving
biomarkers and the NETest. The impartiality of the research report therefor is
not prejudiced." However, Dr. Bodei has on previous papers been listed as
being affiliated with Wren Laboratories. Dr. Oberg, Dr. Krenning, Dr.
Tesselaar, Dr. Ambrosini, Dr. Baum, Dr. Pavel, Dr. Cwikla, Dr. Drozdov, Dr.
Frilling, Dr. Kwekkeboom, Dr. Paganelli, Dr. Severi, Dr. Strosberg, Dr.
Prasad, and Dr. Modlin have been frequent co-authors of NETest papers with
Wren Laboratory researchers. This in no way is to imply that their opinions
are not those of their own, nor to imply any impropriety on their part. This
is not robust support for NETest.

Clinical Utility Assay as a Biomarker for Gastroenteropancreatic and Lung
Neuroendocrine Tumors (25). This is a clinical trial registered in
Clinicaltrials.gov (NCT02948946) with the following Inclusion Criteria:

  * NET Cohort- Patients with histologically or cytologically proven diagnosis of any grade, any stage NET of GEP or lung origin; In the first stage of the study (initial 50 patients) only patients with stage IV, well-differentiated tumors (G1/G2) will be enrolled. 
  * Patients with stable or progressive disease, as documented on a scan (CT, MRI); Progression status will be documented on case report form (CRF). 
  * Allowed prior therapies include: a.) Surgery (tumor surgery at least four weeks prior to study entry); b.) Locoregional therapy such as: chemoembolization, radio-embolization, radiofrequency ablation, radiotherapy at least six weeks prior to study entry; c.) Any number of previous lines of systemic therapy, providing that cytotoxic therapies (chemotherapy, PRRT) have been discontinued at least 4 weeks prior to study entry. 

Non-NET Cohort -

  * Healthy participants
  * Patients with histologically or cytologically proven diagnosis of any grade, any stage GI malignancies. 

Procedure: NETest 5 mL of blood will be drawn from participants for testing.

Estimated Study Completion Date: December 2018

The purpose of this study is to evaluate how well an investigational blood
test performs. The study will look at the sensitivity and specificity of a
blood-based multitranscriptome assay (NETest). Its purpose is not to evaluate
outcomes. This has previously been submitted and reviewed by this contractor
in a prior reconsideration request. At present this is not supportive evidence
for NETest.

Modlin et al. (26) This is a study of 179 cases with 81 gastrointestinal
tumors (41 neuroendocrine tumors of small bowel and 40 being adenocarcinomas
[4 esophageal, 3 gastric, 6 small bowel, 11 colon, 13 rectal, and 3 anal] of
and 98 pancreatic disease (45 known neuroendocrine tumors and 53 patients with
"pancreatic disease") were prospectively collected and assessed using the
NETest and chromogranin A (CgA) to determine metrics for detecting small
intestinal and pancreatic NETs. The term "pancreatic disease" is confusing
since 4 were pancreatic, 31 were pancreatic cysts, 14 were pancreatic
adenocarcinoma, and there were four with neuroendocrine tumors but not
considered with the previous 45 neuroendocrine tumors. Of note, only 35 of the
45 pancreatic neuroendocrine tumors had pathological data. The NETest did
perform significantly better than chromogranin A. However, Dr. Modlin, Dr.
Kidd, Dr. Drozdov, listed under Conflict Of Interest their relationships with
Wren Laboratories, and that "Financial Support: This work was in part
supported by Clifton Life Sciences" which is the parent company of Wren
Laboratories. In addition, nothing in the study shows that the results of the
testing had any change in the outcomes of the patients. This paper has been
previously reviewed by this contractor in a prior reconsideration request.
This is not robust support for NETest.

Modlin et al. (27) This is a single case report concerning neuroendocrine
tumor testing with NETest and chromogranin A, as well as with imaging. As with
all the other Modlin/Kidd papers, this suffers from their association with
Wren Laboratories. In addition, this paper does not show any effect on long
term survival. This paper has been previously reviewed by this contractor in a
prior reconsideration request. This is not robust support for NETest.

Clift et al. (28) Comparison of 3 Systems. This multicenter European study
compared calculated percentage estimates for 5- and 10-year disease-specific
survival using three different prognostic systems in 70 patients with small
bowel neuroendocrine tumors: 1) NET nomogram, 2) the World Health Organization
(WHO)/European Neuroendocrine Tumour Society (ENETS) grading system and 3) the
American Joint Commission on Cancer (AJCC)/ Union Internationale Contre le
Cancer (UICC) TNM staging method. This paper concluded "the NET nomogram may
present a viable alternative to these 'classical' systems as it includes
multiple different aspects of neuroendocrine disease biology specific to each
patient." This is a paper that tries to predict survival. As with all the
other Modlin/Kidd papers, this suffers from their association with Wren
Laboratories. In addition, this paper does not show any effect on long term
survival. This paper has been previously reviewed by this contractor. This is
not robust support for NETest.

Modlin et al. (29) A multianalyte PCR blood test outperforms single analyte
ELISAs (chromogranin A, pancreastatin, neurokinin A) for neuroendocrine tumor
detection. This paper from Wren Laboratories, funded by Clifton Life Sciences,
the parent company of Wren Laboratories, compared PCR-based 51 transcript
signature (known commercially as NETest) and compared it to chromogranin A
(CgA), pancreastatin (PST) and neurokinin A (NKA) testing. Initially there was
a validation set of 40 neuroendocrine tumors and controls and then a
prospectively collected group of 41 neuroendocrine tumors.

Pancreastatin is a derivative of chromogranin A but typically is not used as a
neuroendocrine tumor test. Neurokinin A (NKA, substance K) is a ten amino acid
peptide translated from the pre-protachykinin gene but also not typically used
as a neuroendocrine tumor test. This paper again suffers from much potential
conflict of interest. This paper has been previously reviewed by this
contractor in a prior reconsideration request. This is not robust support for
NETest.

Aluri et al. (3) This is a detailed review of the current status of
biochemical testing in patients with neuroendocrine tumors. The authors note
many of the same problems with testing for these tumors including "the
neuroendocrine tumors (NET) may be functional (secreting 1 or more products
associated with a clinical syndrome) or nonfunctional. Nonfunctional tumors
either fail to secrete any known product, or may secrete a product with no
known associated clinical outcome." They note that clinical presentation may
vary depending on the site of origin of the primary tumor.

They categorize as follows:

**Functional** | **Local symptoms/incidental** | **CgA, (serotonin), PP**  
---|---|---  
**Neuroendocrine tumor sites of origin, potential associated symptoms, and
specific biomarker tests** |  
**Primary Tumor Location** | **Symptom** | **Test**  
**Bronchopulmonary and thymic** | **Local symptoms  
** **Flushing, wheezing  
Cushing syndrome ** | **CgA, serotonin,5-HIAA, 5 hydrosytryptophan  
ACTH, cortisol**  
**Jejunoileal** | **Local symptoms  
Carcinoid syndrome** | **Sertonin  
CgA  
Pancreastatin  
NKA**  
**Colorectal** | **Local symptoms  
Incidental findings ** | **CgA**  
**Pancreaticoduodenal**  
**Nonfunctional** | **Local symptoms/incidental** | **CgA, (serotonin), PP**  
**Functional** | **Specific syndrome** | **Varies per syndrome**  
  
_Abbreviations: 5-HIAA, 5-hydroxyindoleacetic acid; ACTH, adrenocorticotrophic
hormone; CgA, chromogranin A; neurokinin A; PP, pancreatic polypeptide.  _

They caution that all tumor markers have potential for false-positive and
false-negative results.

The authors note, "Because of the difficulties of choosing an optimal
biomarker, along with the interpretation issues of each test, professional
societies have not provided strong recommendations related to minimal
biochemical testing. In general, guidelines from expert committees (European
and American NET societies and the National Comprehensive Cancer Network)
suggest consideration of CgA for small intestinal, nonfunctional pancreatic,
pulmonary, high-grade GEP-NET, and metastases of unknown origin. A 24-hour
urine 5-hydroxyindoleacetic acid test could be considered for small intestinal
and pulmonary NET and for all tumors with carcinoid syndrome. Pancreatic
polypeptide is recommended for nonfunctional pancreatic NET, and neuron-
specific enolase can be considered for high-grade tumors. Specific markers to
assess functionality in patients with syndromes should be guided by the
specific symptoms (eg, urine or plasma 5-hydroxyindoleacetic acid for
carcinoid syndrome, insulin for hypoglycemia, etc). Our own practice is to
test a broad range of markers initially and then follow markers that are
positive."

The authors specifically mention the NETest in detail. "There have been recent
developments in nucleic acid-based technologies, like microRNA profiling, and
strategies to collect circulating tumor cells, which may become helpful in
future for NET diagnosis and management. At this time, however, the only
commercially available multianalyte NET test, which may be clinically helpful,
is the NETest. The NETest, developed by Wren Laboratories (Branford, CT), is a
multianalyte quantitative reverse-transcriptase polymerase chain reaction
assay, based on 51 marker genes with an algorithmic analysis, claims a high
sensitivity (>95%) and specificity (>95%) in the detection of all GEP-NET
tumors. The test offers an assessment of disease status and treatment
effectiveness. Early data suggest that the NETest may be more accurate than
single biomarker or monoanalyte tests and it is not known to be affected by
age, gender, fasting status, or use of proton pump inhibitors. The test
requires a single laboratory, Wren Laboratories, to offer the specialized
analysis. More information is needed on whether the test is positive in non-
GEP-NET or cancers with mixed epithelial and NET phenotype, for example,
prostate cancer. Additionally, more prospective verification studies by
investigators independent of the manufacturing company are needed. At this
time, there is no specific recommendation from any professional organization
supporting the NETest." The article notes, "The authors have nothing to
disclose." Thus while this article offers positive comments about the NETest,
it does not support its use currently.

Peczkowska et al. (30) This study assessed the proprietary 51-neuroendocrine
gene blood analysis (NETest) in 32 well-differentiated paragangliomas and
pheochromocytomas compared to chromogranin A (CgA) and catecholamine
measurements. A control group (without neuroendocrine tumors) included one
person with inflammatory bowel disease, three with IPMN [not defined by the
authors, but most likely is intraductal papillary mucinous neoplasm], nine
with benign pancreatic cysts and 19 otherwise healthy controls. The results of
NETest were 100% positive for those with neuroendocrine tumor and negative for
the controls. Again, there is much conflict of interest and no evidence of
improved outcomes. This paper has been previously reviewed by this contractor
in a prior reconsideration request. This is not robust support for NETest.

Verbeek et al. (31) This is a review of neuroendocrine tumors (NET), including
their presentation, location in the body, and associated biomarkers, including
Chromogranin A (CgA)) and NETest. The summary is, "NETs are rare tumours, with
an annual incidence estimated as 2-5 cases per 100,000 population. They are
able to produce non-specific and specific biomarkers. Presently the most
frequently used biomarker in GEPNETs [gastrointestinal or pancreatic origin"
is CgA, although it is a non-specific biomarker and has its limitations. More
specific markers can be used in functioning NET. The use of NEN
[neuroendocrine neoplasms] transcripts is promising, but its application of
this PCR-based blood test in patients with a NET is still in research." Unlike
all the other NETest publications, this one is not affiliated with Wren
Laboratories or Clifton Life Sciences. This study also has no evidence of
improved outcomes. This paper has been previously reviewed by this contractor
in a prior reconsideration request. It is not robust support of NETest.

Bodei et al. (32) Forty-nine patients with neuroendocrine tumors were assessed
for the concordance between Ga-somatostatin analog (SSA) positron emission
tomography (PET), circulating NET gene transcripts (NETest), chromogranin A
(CgA), and the antigen Ki-67. The authors concluded "Ga-SSA PET imaging
parameters (SUVmax) [the maximum standardized uptake value] correlated with a
circulating NET transcript signature. Disease status could be predicted by an
elevated quotient of gene expression (MORF4L2) and SUVmax. These observations
provide the basis for further exploration of strategies that combine imaging
parameters and disease-specific molecular data for the improvement of NET
management. Of note, Dr. Kidd, Dr. Modlin, and Dr. Drozdov are listed as being
from Wren laboratories, " but reported "Conflicts of interest: None." This
study also has no evidence of improved outcomes. This paper has been
previously reviewed by this contractor in a prior reconsideration request. It
is not robust support of NETest.

Bodei et al. (33) Seventy-eight patients with neuroendocrine tumors were
treated with Peptide Receptor Radionuclide Therapy (PRRT) using 177LuDOTA-
Tyr3-Thr8-octreotide (177Lu-octreotate). However, the paper only analyzed
fifty-four patients as an interim report. There was definitive data on 60
patients, but three were excluded because of withdrawal of consent, two died,
and one was missing data before dropping out due to progression. The study was
designed to assess whether a circulating multianalyte 51-gene NET signature
(NETest), compared to chromogranin A (CgA), could be used as a surrogate
measure of clinical responses to PRRT when assessed at 3- and 6-months follow-
up. There was a 72% disease control rate, although this determination is
difficult to understand. Changes in NETest accurately (89 %) correlated with
treatment response, while chromogranin A (CgA) was only 24 % accurate.
However, the paper did conclude, "Confirmation of these observations in larger
series will allow identification of likely non-responders and will better
define at a molecular level the natural history of individual neuroendocrine
tumors." Dr. Kidd listed himself as from Wren Laboratories, although Dr.
Modlin did not. "Disclosure of potential conflicts of interest: The authors
declare that they have no conflict of interest." This study also has no
evidence of improved outcomes. This paper has been previously reviewed by this
contractor in a prior reconsideration request. It is not robust support of
NETest.

Cwikla et al. (34) This short study (median 10 months) and relatively small
study of 35 patients with neuroendocrine tumors reports on patients who were
treated with somatostatin analogs (octreotide or pasireotide) and who were
followed by NETest and Chromogranin A (CgA) testing. These patients had known
disease status of either stable or progressive. There also was a prospective
set of 28 patients who were treated with somatostatin analogs (octreotide or
lanreotide). In the test set, the NETest was positive in all patients. Twenty-
five (71%) were classified as stable disease by imaging. The NETest activity
was significantly lower in stable disease than in progressive disease treated
with somatostatin analogs. The twenty-eight patients in the prospective
assessment set were evaluated. They were on treatment with somatostatin
analogs, although 50% were not on the same ones as the other set. In addition,
the description of the differences between the two sets is a bit confusing.
The NETest was positive in all patients and the mean levels significantly high
in the group that subsequently developed progressive disease. However,
fourteen with an elevated NETest of 80-100% during the course of treatment
developed progressive disease, but six with these elevated did not develop
progressive disease. Chromogranin A (CgA) elevation of greater or equal to 25%
may have indicated progressive disease. Dr. Kidd is from Wren Laboratories and
the work was supported by Clifton Life Sciences, the parent company of Wren
Laboratories. However, the "Disclosure summary" was "The authors have nothing
to disclose." There is no evidence that this testing affected outcomes. This
paper has been previously reviewed by this contractor in a prior
reconsideration request. It is not robust support of NETest.

Pavel et al. (35) This small study (n=34 originally but three were excluded in
the follow up) followed patients with gastroenteropancreatic endocrine tumors
for a median of 4 years (range 2.2-5.4 years). Seventeen were grade 1, 14
grade 2, and 1 grade 3 (2 had no grade available). Thirty-one were stage IV.
They were evaluated using the NETest and chromogranin A (CgA) testing. This
was not a prospective study and not a homogenous group. Some of the data were
extracted and confirmed retrospectively. At baseline all the NETest were
positive and the chromogranin A (CgA) were elevated in 50%. The evaluation is
somewhat confusing since three were excluded from the group analysis. The
Kaplan-Meier Analysis of T0 to first restaging however, was not significant
for the NETest. Overall, the NETest was better than the CgA testing at
following patients. The "Disclosure Statement" was, "There is no conflict of
interest." However, "The study was supported by Clifton Life Sciences" which
is the parent company of Wren Laboratories, Inc. Also, Dr. Drozdov, Dr.
Modlin, and Dr. Kidd were listed as being from Wren Laboratories. There is no
evidence that this testing affected outcomes. This paper has been previously
reviewed by this contractor in a prior reconsideration request. It is not
robust support of NETest.

Modlin et al. (36) Thirty five patients with gastroenteropancreatic
neuroendocrine tumor were studied. Twenty-seven were operated on: twenty-one
to remove the primary tumor and local/regional lymph nodes; two for debulking;
three for suspicion of neuroendocrine tumor of the small bowel; and one for
appendectomy. It is unknown if the appendix patient had a localized,
incidental neuroendocrine tumor. Eight others received non-operative
strategies including embolization (three), trans-arterial chemoembolization
(two), and radiofrequency ablation (three). Preoperative blood samples were
collected (0-24 hours before treatment) and 1 month post-therapy. NETest and
chromogranin A (CgA) testing were done. Follow-up including imaging including
CT scan, magnetic resonance imaging (MRI), and
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-PET (United Kingdom
alone) was performed on all patients at three and six months. For the entire
operative cohort (n = 27), the pre-surgery NETest scores were increased (79.8
 5.1%). Twenty-three (85%) of the 27 subjects exhibited a decreased score
post-surgery (4 patients were unchanged). CgA levels were normal in 18 (67%)
of the 27 subjects before surgery and in 3 (40%) of 8 subjects before
ablation. The postoperative levels of the 2 groups were not significantly
different. The post-ablation levels were not reduced. Overall, 6 (22%) of the
27 subjects exhibited a decrease in CgA levels post-surgery. Of the 9 patients
with increased preoperative CgA levels, six (67%) exhibited a normalization of
values after surgery. The article concluded, "Our results suggest that a PCR-
based blood test will be useful in the assessment of the adequacy of operative
resection, but a further long-term prospective study is needed to establish
the most accurate timing of blood collection (postsurgery) as well as the
metrics of the NETest in the prediction of residual/recurrent disease. Under
such circumstances, the role of adjuvant therapy may, in the future, become a
relevant consideration for NET patients with R0 ["absence of tumor after
surgical treatment"] resected disease." There was no conflict of interest
disclosure at all. However, Ms. Alaimo, Mr. Callahan, Ms. Teixeira, Dr. Bodei,
Dr. Drozdov, and Dr. Kidd were noted to represent Wren Laboratories, the maker
of NETest. There is no evidence that this testing affected outcomes. This
paper has been previously reviewed by this contractor in a prior
reconsideration request. It is not robust support of NETest.

Kidd et al. (37) This was submitted in a recent reconsideration request to
this contractor and is being reviewed as a new reference. The authors
evaluated whether the NETest identified bronchopulmonary neuroendocrine tumors
in 194 samples including 65 controls, 14 benign lung disease, 25
bronchopulmonary neuroendocrine tumors, 25 small bowel neuroendocrine tumors,
36 lung adenocarcinomas and 29 lung squamous cell carcinomas had blood collect
immediately prior to surgery or at regular follow-up or controls. Then in
seven surgical resected bronchopulmonary neuroendocrine tumors, they compared
the tissue NETest with serum testing. They concluded that there was
significant correlations between the seven paired tumors and blood samples.
Also, there were significant differences in NETest results between lung
cancers and benign diseases. In addition gene expression was highly correlated
between small bowel and bronchopulmonary neuroendocrine tumors. The paper
concluded, "CONFLICTS OF INTEREST None" although Dr. Kidd, Dr. Drozdov, Dr.
Matar, and Dr. Chung were from Wren Laboratories. No affiliation for Dr.
Modlin was mentioned with Wren Laboratories. Again, there was no evidence that
this testing would lead to increased survival. It is not robust support of
NETest.

Filosso et al. (38) This was submitted in a recent reconsideration request to
this contractor and is being reviewed as a new reference. The authors
evaluated their NET multigene blood test (NETest) to diagnose bronchopulmonary
neuroendocrine tumours, assess disease status and evaluate surgical resection.
This multicenter retrospective diagnostic study of 118 patients who had
histological confirmation of bronchopulmonary carcinoids. There were 90
healthy controls, 18 people with chronic obstructive pulmonary disease, 13
"other neuroendocrine neoplasms" (9 large cell neuroendocrine carcinoma and 4
small-cell lung carcinomas), 23 squamous cell lung carcinomas, 26 lung
adenocarcinomas. Seventy-four of the carcinoids were stable, 38 had
progressive disease and 6 were surgical cures as defined as disease free.
Blood testing for NETest and chromogranin A (CgA) were both performed on the
samples. The NETest was positive in all the bronchopulmonary carcinoids. The
chromogranin A was elevated in 44 of them. There were 28 patients who
underwent surgery (19 with lung neuroendocrine neoplasm) and nine described as
"lung cancers". The patients with neuroendocrine lung, NETest were
significantly reduced following surgery. There was no significant alternation
in values noted in the lung cancer cohort. The chromogranin A was not
significantly reduced by surgery. While much of the study was retrospective,
and the prospective number was small, this does support that NETest is more
accurate than chromogranin A. However this work was supported by the Clifton
Life Sciences. In addition Dr. Kidd, Dr. Chung, and Dr. Drozdov are listed as
being from Wren Laboratories. Despite of this, and the fact that Dr. Modlin
has been on other papers been affiliated with Wren Laboratories, "Conflict of
interest: none declared" was stated. No evidence was submitted that this led
to increased survival. It is not robust support of NETest.

Tsoukalas et al. (4) This is a 2018 "Review Article on Breakthroughs in the
Treatment of Advanced Lung Cancer: Progress Through Innovation." It reviews
the most recent surgical treatments, medical treatments, molecular-targeted
therapies for bronchopulmonary neuroendocrine tumors. There is no mention of
NETest. The "Conflicts of Interest: The authors have no conflicts of interest
to declare." None of the authors are listed as associated with any proprietary
company. It is not support of NETest.

Howe et al. (39) The authors note, "Not surprisingly, there has been much
confusion and controversy surrounding the management of patients with SBNETs
[small bowel neuroendocrine tumors], and there are no randomized studies that
define their optimal surgical treatment. Therefore, in treating these
patients, clinicians must rely on their experience and the results of
retrospective studies, both of which are subject to bias. Furthermore, there
may be significant differences in opinion among the physicians taking care of
these patients, depending on whether they are surgical oncologists, medical
oncologists, endocrinologists, gastroenterologists, interventional
radiologists, or nuclear medicine physicians. Both the European Neuroendocrine
Tumor Society (ENETS) and North American Neuroendocrine Tumor Society (NANETS)
have published consensus guidelines for the diagnosis and management of
SBNETs, but there remain many clinical scenarios for which the ideal approach
is unclear." This consensus guideline from the North American Neuroendocrine
Tumor Society does mention chromogranin A and other testing, but not NETest.
It does mention several articles by Modlin and Kidd, but none pertained to the
NETest. It is not support of NETest.

Hope et al. (40) This paper evaluates the somatostatin receptor positron
emission tomography (SSTR-PET) for imaging in patients with neuroendocrine
tumors and that it has demonstrated a significant improvement over
conventional imaging. They recommend that SSTR-PET should replace In-111
pentetreotide scintigraphy (OctreoScan) in all indications in which SSTR
scintigraphy is currently being use. The paper includes one reference from Dr.
Modlin, but it is a 2010 one entitled "Pathologic Classification of
Neuroendocrine Tumors: a Review of Nomenclature, Grading, and Staging
Systems." There is no mention of NETest and thus does not support NETest.

Strosberg et al. (41) This is a review article on midgut neuroendocrine
tumors. In particular, monitoring and markers are discussed. A significant
majority of the expert panel indicated that they routinely monitor serotonin
output in patients with advanced midgut NETs, typically at the time of
radiographic staging. There was no consensus regarding the optimal method for
measurement of serotonin output including 5-HIAA. Concerning monitoring of
nonhormonal tumor markers in patients with advanced midgut NETs, they note,
"Chromogranin A is the most commonly measured nonspecific tumor marker in
patients with midgut NETs; however, there was consensus that high rates of
false-positive and false-negative results as well as unexplained fluctuations
limit its utility. Chromogranin A has been validated as a prognostic marker in
midgut NET in randomized clinical trials. Pancreastatin, a breakdown product
of CgA, may be more specific in certain contexts, such as patients using
proton pump inhibitors (which raise CgA levels). A significant majority of the
expert panel reported that they measure tumor markers such as CgA and/or
pancreastatin in routine practice, but a significant majority also indicated
that these tumor markers assist in patient management only occasionally or
rarely. As a result, no consensus was achieved on whether tumor markers should
be routinely measured in patients with advanced midgut NETs. Studies of the
relatively novel 51-gene, polymerase chain reaction-based NETest report higher
rates of sensitivity specificity, and accuracy compared with conventional
monoanalyte tumor markers. Validation studies are ongoing." Only one person,
Dr. Halfdanarson, is listed as having any relationship with Clifton
Biosciences, although previously, Dr. Strosberg is noted to have a financial
relationship with Wren Laboratories. No evidence was submitted that this led
to increased survival. It is not robust support of NETest.

Lipinski et al. (42) This is an overall review of the topic of gastroduodenal
neuroendocrine neoplasms. While serum chromogranin A (CgA) is recommended,
there is no discussion of NETest. There is no statement of conflict of
interest. This does not support NETest.

Bednarczuk et al. (43) This is an overall review of the topic of
neuroendocrine neoplasms of the small intestine and appendix. It states that
"The most useful laboratory test is the determination of chromogranin A, while
concentration of 5-hydroxyindoleacetic acid is helpful in the diagnostics of
carcinoid syndrome." There is no discussion of NETest. There is no statement
of conflict of interest. This does not support NETest.

Starzynska et al. (44) This is an overall review of the topic of colorectal
neuroendocrine tumors/neoplasms. It states that **"** there is a growing body
of evidence supporting the thesis that the neuroendocrine neoplasms of the
rectum and the neuroendocrine neoplasms of the colon are two different
diseases." There is no mention of any biomarkers. There is no discussion of
NETest. There is no statement of conflict of interest. This does not support
NETest.

Saif MW. (2) In this review article, the author notes, "Targeted therapies
such as everolimus and sunitinib resulted in statistically significant
improvements in PFS [progression free survival] in randomized studies, and can
be used in well to moderately differentiated metastatic pNETs (pancreatic
neuroendocrine tumors). However, they are rarely associated with objective
radiographic tumor shrinkage by Response Evaluation Criteria in Solid Tumors
(RECIST) and the response rates associated with these agents are quite low as
well. The choice between everolimus and sunitinib is difficult as they have
very similar improvements in PFS [progression free survival] in phase III
clinical trials. One major criticism of the results of both phase III studies
would be that significant improvement of PFS [progression free survival] is
just a surrogate marker, since significant prolongation of OS [overall
survival] has not been shown. However, the design of a phase III study with OS
[overall survival] as primary endpoint and a study power of 90% would require
an estimated sample size of 2800 patients in order to show that a survival
benefit of 4 months is significant."

He also notes, "Biomarkers may be used to assist in both the diagnosis and
post-treatment follow up in patients with pNETs (pancreatic neuroendocrine
tumors). In poorly differentiated tumors, chromogranin-A (CgA), synaptophysin
and cytokeratin are often used to establish neuroendocrine differentiation.
Ki-67 (MB-1) and the mitotic rate are used as grading criteria for NETs and
were shown to correlate with outcome. Blood markers, such as pancreatic
polypeptide (PPP) and specific serum hormone levels are also used to diagnose
pNETs (pancreatic neuroendocrine tumors) and evaluate response to therapy.
Plasma levels of chromogranin-A are thought to correlate with tumor burden and
increased levels have been associated with a poor PFS and survival [24]. PPP
[pancreatic polypeptide] is a nonspecific biomarker for nonfunctioning pNETs
(pancreatic neuroendocrine tumors) with a relatively low sensitivity (63%) and
specificity (81%). However, when used in combination with CgA, the sensitivity
for nonfunctioning pNETs (pancreatic neuroendocrine tumors) increased from 68%
to 93%. For functioning pNETs (pancreatic neuroendocrine tumors), the levels
of the secreted hormone represent a more specific tumor marker. As per the
National Comprehensive Cancer Network (NCCN) guidelines, serum proinsulin,
insulin/glucose ratio and C-peptide levels may be followed in insulinomas;
serum gastrin levels may be followed in gastrinomas; serum VIP levels may be
followed in VIPomas; serum glucagon, glucose and CBCs may be followed in
glucagonomas; serum Somatostatin, calcitonin and parathyroid hormone related
peptide levels may be followed in other functioning pNETs (pancreatic
neuroendocrine tumors). Novel biomarkers are currently being evaluated,
including neuron-specific enolase (NSE), circulating tumor cells (CTCs), and
placental growth factor. Currently, no definitively proven predictive
biomarkers are available to guide selection of therapies. Therefore, further
studies are needed to individualize and optimize their management. Over the
past few years, knowledge regarding the molecular pathology of Pancreatic NETs
has increased substantially. PNETs (pancreatic neuroendocrine tumors) are
characterized by a relatively limited number of mutations in tumor suppressor
genes, such as MEN1, ATRX and DAXX. The clinical significance and potential
for treatment of these mutations remains uncertain." There is no mention of
NETest or Dr. Kidd or Dr. Modlin in this review article. Most important, it
specifically noted, "significant improvement of PFS [progression free
survival] is just a surrogate marker, since significant prolongation of OS
[overall survival] has not been shown." This does not support NETest.

Koenig et al. (7) This paper reviewed 62 individuals treated at the University
Hospital of Marburg in Germany. The paper also reviews the neuroendocrine
tumors and states "chromogranin A (CgA) is a well-established marker for
diagnosis and follow up of patients with gastroenteropancreatic neuroendocrine
neoplasms" and "it has been shown that plasma levels of chromogranin A
correlate with tumor load and predict survival of patients with neuroendocrine
neoplasms of the small bowel." They state that this study "demonstrates that,
immunohistochemical CgA (chromogranin A) and synaptophysin are good markers
for histological diagnosis in patients with NEN (neuroendocrine neoplasms) of
the colon and rectum. However, chromogranin A is a poor marker to follow-up
these patients because only a minority exhibited increased levels which did
not increase significantly during tumor progression. In contrast to NEN
(neuroendocrine neoplasms) of the small bowel, there is no correlation of CgA
plasma levels with tumor burden or survival. Patients with NEN (neuroendocrine
neoplasms) of the colon and rectum displayed a relatively good prognosis
resulting in a median survival of 8.5 years. However, a subset of patients
affected by G3 neoplasms, exhibited a poorer prognosis with a median survival
of 2.5 years. Taken together, CgA is a valuable marker for immunohistochemical
diagnosis, but CgA plasma concentration is not suitable to mirror tumor burden
or prognosis in patients with NEN of the colon and rectum." "Usually, NEN are
well-differentiated tumors exhibiting a slow growth associated with a good
prognosis. However, some of these tumors grow more rapidly, resulting in less
favorable survival rates. In fact, there are currently no well-established
prognostic markers for patients with these tumors. Histological staining for
Ki-67, the degree of differentiation of the tumor, the presence of metastases,
or lymph node involvement are currently the most reliable markers to predict
tumor growth and survival of patients with neuroendocrine tumors." It notes
that "currently, CgA is widely used in the clinical routine for diagnosis and
follow-up of patients with gastroenteropancreatic neuroendocrine neoplasms."
The paper did note, "NETest was superior in predicting progressive disease and
disease alterations compared to CgA. However, patients with colorectal NEN
(neuroendocrine neoplasms) were not included in this pilot project and
actually the NETest is not routinely available. Whether the use of a recently
suggested array-based, multifactor analysis of blood mRNA levels can overcome
this problem in daily patient care must be evaluated in future studies." This
is not robust support for NETest.

Bowden et al. (10) As noted above, MiRNAs are short (approximately 22
nucleotides) RNA sequences that have been shown to broadly regulate gene
expression at a post-transcriptional level, by binding to the 3' region of
target RNAs, resulting in mRNA degradation and inhibition of translation.
Using a 742-miRNA panel, the authors identified candidate miRNAs similarly
expressed in 19 small intestine neuroendocrine tumors and matched plasma
samples. They refined their panel in an independent cohort of plasma samples
from 40 patients with metastatic small intestine NET and 40 controls, and then
validated this panel in a second, large cohort of 120 patients with metastatic
small intestine NET and 120 independent controls. The authors concluded that
elevated circulating levels of miR-21-5p and miR-22-3p and low levels of
miR-150-5p are characteristic in patients with metastatic small intestine
neuroendocrine tumors, and further suggests that levels of these miRNAs are
associated with overall survival. These observations provide the basis for
further validation studies, as well as studies to assess the biological
function of these miRNAs in small intestine neuroendocrine tumors. "CONFLICTS
OF INTEREST The authors declared that there are no conflicts of interest in
this work." This paper supports the role measuring miRNA in patients with
neuroendocrine tumors. However, the results of the testing did not affect
survival. In addition, there is nothing to suggest that these are the same
testing that is performed by NETest. The authors acknowledge NETest by noting,
"Preliminary studies have suggested that a multianalyte approach, using a
51-transcript mRNA panel, may be more sensitive and specific than currently
available biomarkers though this assay has yet to be validated in large,
prospective studies." This paper has been previously reviewed by this
contractor in a prior reconsideration request. This is not robust support for
NETest.

Delle Fave et al. (45) This European Neuroendocrine Tumor Society (ENETS)
guideline gives a current overview on the histology, classification, and
treatments, but does not discuss testing, or follow-up surveillance, outside
of endoscopy. There are no disclaimers or conflict of interest mentioned. This
is not supportive of NETest.

Niederle et al. (46) This European Neuroendocrine Tumor Society (ENETS)
guideline deals only with non-metastatic neuroendocrine neoplasms originating
from the small bowel. It does note, "Serum chromogranin A (CgA) remains a
relatively sensitive marker for NENs (neuroendocrine neoplasms) of all origins
including Si-NENs (small intestine neuroendocrine neoplasms). CgA has also
more recently been shown to prognostically predict significantly differing
groups, with higher levels of CgA indicating a worse prognosis, probably
related to increased tumor cell mass. For longitudinal follow-up purposes, it
is important to note that absolute CgA values may differ significantly between
different assays and therefore it is recommended to perform repeated
measurements in the same laboratory or at least with the same assay whenever
possible. Furthermore, the differential diagnosis of elevated CgA values such
as in patients on proton pump inhibitors (PPIs), with chronic atrophic
gastritis, chronic renal failure, liver cirrhosis or congestive heart failure,
as well as other CgA-secreting neoplasms (e.g. hepatocellular carcinoma,
medullary thyroid carcinoma) needs to be considered when CgA values are
interpreted. CgA may signal NEN (neuroendocrine neoplasms) recurrence after
successful curative resection early in patients with a small tumor burden."

"Endocrine tumors of the jejuno-ileum produce serotonin and elevated 24-hour
urinary 5-hydroxy indole acetic acid (5-HIAA) levels as a product of the
metabolism of serotonin. 5-HIAA has a sensitivity of up to 100% and a
specificity of 85-90% for detecting a carcinoid syndrome, and a sensitivity of
70-75% and a specificity of close to 100% for predicting a primary tumor in
the jejuno-ileum. Urinary 5-HIAA should be collected with strict dietary
restrictions to avoid false positive levels. Serum serotonin determinations
are less sensitive and specific and are, therefore, not recommended; serotonin
measurements in platelets, where serotonin is stored depending on its
availability in the systemic circulation, may be even more sensitive, but is
not widely available and therefore currently impractical."

"Minimal Consensus Statement on Laboratory Tests: The minimally required
biochemical tests include plasma CgA and urinary 5-HIAA. These tests should be
performed at the first visit and then for follow-up or on suspicion of NEN
recurrence or progression. Newer markers, either biochemical or based on
circulating NEN cells, require further validation. Neuron- specific enolase
has no role for the diagnosis of these almost always well- to moderately
differentiated NEN (G1/2 NET)." There are no disclaimers or conflict of
interest mentioned. This is not supportive of NETest.

Crabtree JS. (9) This review paper, cited above, referenced three articles by
Dr. Modlin, but concluded, "Furthermore, genetics-based approaches may hold
the key toward precision therapies and future investigations into novel
pathways may help define driver mutations present in the different subtypes of
NETs. Future clinical utilization of gene panels, methylation screening tools
and other molecular biomarker approaches in addition to classical
neuroendocrine markers, will facilitate treatment and improve outcomes of this
disease." This work was supported by the Louisiana State University Health
Sciences Center Department of Genetics. No mention of conflict of interest was
stated. This does not support NETest.

Childs et al. (47) These authors note, that other biomarkers for evaluating
neuroendocrine tumors include overexpressed somatostatin receptors (SSTR) that
can be targeted for therapy. Somatostatin receptors 2 and 5 are detectable on
CTCs from NET patients and may be a useful biomarker for evaluating SSTR-
targeted therapies and this is being prospectively evaluated in the Phase IV
CALMNET trial (NCT02075606). "Conflict of Interest: Dr. Meyer received
research funding for this work from Ipsen and Dr. Caplin has received grant
funding and consultancy fees from Ipsen." In addition, "The funding was
provided by the University College London (UCL) Experimental Cancer Medicine
Centre Grant No. C12125/A15576, the UCL Hospitals NIHR Biomedical Research
Centre and Ipsen." This does not support NETest.

Kunz PL. (48) This is a commentary about the Dasari article. It deals with
increasing incidents of neuroendocrine tumors and the "need to conduct
rigorous basic, translational, clinical, and health services research to move
the field forward." NETest is not discussed, although neither are other
markers. It does not support, nor refute NETest.

Berardi et al. (49) This is a detailed review on neuroendocrine tumors (NET)
and treatment methods. The authors note, "Therapeutic sequence is still
controversial. To date research efforts are focused on translational studies
on the available options in order to better select the patients who could
benefit from different treatment options. In fact a better understanding of
the molecular pathways involved in the NETs carcinogenesis, might predict the
efficacy of each therapeutic approach. Furthermore, research on identifying
the best strategy involving the available options would be beneficial."
"Conflict of interest: All authors disclose no financial and personal
relationships with other people or organizations that could inappropriately
influence (bias) their work." "Acknowledgement: The authors did not receive
specific funding for the manuscript." This paper does not support NETest.

Kwon et al. (50) This is a review article that brings up a very important
point. "The prevalence of gastric NETs is difficult to establish due to a lack
of uniform data collection from cancer registries worldwide. A study published
in 2015 analyzing national cancer registries in 10 European countries, the US,
and Japan determined the prevalence of gastric NETs per 10,000 population to
be 0.32 in Europe, 0.17 in the US, and 0.05 in Japan. The authors of this
study did note that their values may be underestimations due to a tendency of
cancer registries to reflect the aggressive tumors that require treatment as
opposed to benign tumors." While this paper does cite an article by Dr. Kidd
and Dr. Modlin, no biomarkers except gastrin are mentioned. "Disclosure: EKB
has received research funding (to institution) from Novartis and served as a
consultant (unpaid) for Ipsen. The other authors report no conflicts of
interest in this work." This does not support NETest.

Oronsky et al. (51) This is a review article about neuroendocrine tumors
including history, workup, and treatment. The article notes, "Commonly
measured tumor markers in NENs include serum CgA and 5-HIAA, the final
secreted product of serotonin, levels in a 24-hour urine sample. Since serum
CgA, which is a more sensitive and broadly applicable marker than urinary 5
**-** HIAA, does not depend on serotonin secretion, it is preferred over
5-HIAA for bronchial and rectal tumors, which do not generally secrete
serotonin. In addition to its value in making the diagnosis, plasma CgA levels
correlate with tumor bulk, differentiation and secretory activity, which in
turn may predict treatment response and overall survival (OS) (fast rising
levels seem to indicate a poor prognosis). Besides CgA and 5-HIAA, NENs are
known to produce a plethora of bioactive amines and peptides such as
5-hydroxytryptamine, 5-hydroxytryptophan, serotonin, insulin, gastrin,
glucagon, somatostatin, vasoactive intestinal peptide, growth hormone,
adrenocorticotropic hormone, melanocyte-stimulating hormone, pancreatic
polypeptide, calcitonin, substance P, pancreastatin, etc., resulting in
relatively uncommon but unique clinical syndromes." A concern is "Funding:
None." and no conflict of interest statement. This is a bit concerning since
Dr. Oronsky is from EpicentRx Inc, a pharmaceutical company involved in cancer
research. Of note, the paper references Dr. Modlin multiple times, but not for
NETest. This is not a supportive paper of NETest.

Falconi et al. (52) This international (European Neuroendocrine Tumor Society)
consensus guideline does not mention NETest. This is not a supportive paper of
NETest.

Garcia-Carbonero et al. (53) This international (European Neuroendocrine Tumor
Society) consensus guideline does not mention NETest although it mentions
chromogranin A. There are no disclaimers or conflict of interest mentioned.
This is not a supportive paper of NETest.

Kaltsas et al. (54) This international (European Neuroendocrine Tumor Society)
consensus guidelines on pre-and perioperative therapy in patients with
neuroendocrine tumors does not mention NETest although it mentions
chromogranin A. There are no disclaimers or conflict of interest mentioned.
This is not a supportive paper of NETest.

Pavel et al. (55) This international (European Neuroendocrine Tumor Society)
consensus guidelines for the standards of care in neuroendocrine tumors does
not mention NETest. This paper has been previously reviewed by this contractor
in a prior reconsideration request. It is the introductory article for the
following series of articles. There are no disclaimers or conflict of interest
mentioned. This is not a supportive paper of NETest.

Perren et al. (56) This international (European Neuroendocrine Tumor Society)
consensus guidelines for standards of care in neuroendocrine tumors and their
pathological diagnosis and prognostic stratification does not mention NETest.
It does mention and support chromogranin-A. It does conclude, "translational
studies are needed to define biomarkers predicting response to other therapies
such as targeted therapies or other chemotherapeutic strategies. **" **This is
not a supportive paper of NETest.

Sundin et al. (57) This international (European Neuroendocrine Tumor Society)
consensus guidelines for standards of care in neuroendocrine tumors and their
radiological imaging does not discuss biomarkers. It is included in this
review for completeness. It does not support, nor refute NETest.

Oberg et al. (58) This international (European Neuroendocrine Tumor Society)
consensus guidelines is completely devoted to biomarkers in neuroendocrine
tumors. It goes into great depth on the various biomarkers. In particular, it
notes "Overall CgA [chromogranin-A] has been found to be clinically
informative and moderately sensitive in the majority of studies devoted to
this topic. It does note the false positives associated with chromogranin-A.
It notes "A recognized international standard for CgA assay is not available
and variations in assay types may influence results. Several assays for
measurements of intact CgA and cleavage products have been developed"
Commercial CgA kits differ in the types of antibodies used (monoclonal vs.
polyclonal) and include enzyme detection (ELISA) and radioimmunoassay.
Differences in methods of standardisation have also lead to heterogeneity. In
particular it notes:

  * CgA is the most practical and useful general serum tumor marker in patients with NETs;
  * Elevated CgA can occur in normal individuals and in patients with non-NET tumors although the levels are usually lower than in patients with NETs;
  * Sensitivity of elevated CgA varies according to NET tumor type and volume;
  * Reference laboratories should be preferred for clinical samples assays;
  * Reference intervals and individual patient results differ significantly between different CgA assays and cannot be directly compared;
  * Serial measurements should be performed using the same assay;
  * If assays are changed, patients should undergo a new baseline measurement;
  * False-positive results are possible in patients with hypergastrinemia (especially on antisecretory medications or chronic atrophic gastritis Type A) and in the presence of heterophile antibodies (care in patients with autoimmune diseases or those sensitized to rodent proteins [mouse monoclonal antibodies]);
  * Where possible, PPIs should be interrupted, leaving a clearance of at least 3 half-lives, prior to CgA plasma sampling. 

It does note, "blood measurements of neuroendocrine gene transcripts have
demonstrated significant diagnostic and prognostic potential in recent studies
(NET-test). The precise role of these analyses has to be expected in future
prospective trials" and does cite three Modlin/Kidd publications. This is not
robust support for NETest.

Partelli et al. (59) This international (European Neuroendocrine Tumor
Society) consensus guidelines for standards of care for small intestinal and
pancreatic neuroendocrine tumors does not discuss biomarkers. It is included
in this review for completeness. It does not support, nor refute NETest.

Pavel et al. (60) This is another international (European Neuroendocrine Tumor
Society) consensus guideline on the standards of care concerning systemic
therapy for neuroendocrine neoplasms. It specifically mentions chromogranin-A
and several other biomarkers to be performed before and after treatment, but
does not mention NETesting. There are no disclaimers mentioned. This paper
does not support NETesting.

Garcia-Carbonero et al. (61) This is another international (European
Neuroendocrine Tumor Society) consensus guideline on the standards of care
concerning systemic chemotherapy for neuroendocrine neoplasms. Like the
preceding reference, it specifically mentions chromogranin-A and several other
biomarkers to be performed before and after treatment, but does not mention
NETesting. This paper does not support NETesting.

Hicks et al. (62). This is another international (European Neuroendocrine
Tumor Society) consensus guideline on the standards of care concerning peptide
receptor radionuclide therapy for neuroendocrine neoplasms. It specifically
notes, "the availability of serum biomarkers like chromogranin-A (Cg-A) or
other specific hormones also provides an important means to assess response."
It does mention that chromogranin-A is not always elevated in these metastatic
tumors and can be elevated in other situations. It does state, "there is a
need for improved biomarkers of response." However, NETesting is not mentioned
at all. This paper does not support NETesting.

Modlin et al. (63) This paper is now really a historical paper. It describes
the development of the NETest, noting that "PCR-based signature measures
multiple transcripts which reflect the diverse biological profile of a
proliferating NEN and may, with further examination in appropriate studies, be
tested as a measure of tumor responsiveness and, potentially, as a
prognostic." It was not associated with any improved clinical outcomes. It was
reviewed by this contractor in a prior reconsideration and not felt to
adequately support NETest.

Modlin et al. (64) This paper is now also a historical paper. It describes the
development of the NETest, noting that "These parameters indicate this may
provide an accurate and sensitive multi transcript molecular tool to identify
NENs and assess disease progress using peripheral blood sample." It was not
associated with any improved clinical outcomes. It was reviewed by this
contractor in a prior reconsideration and not felt to adequately support
NETest.

Pavel et al. (65) This paper was reviewed by this contractor in a prior
reconsideration. It is not from Dr. Modlin or Dr. Kidd. The study does not
address NETest and thus does not support the use of NETest.

Caplin et al. (66) This paper was reviewed by this contractor in a prior
reconsideration. It is an excellent paper in the _prestigious New England
Journal of Medicine_ concerning the somatostatin analogue lanreotide in
patients with advanced, well-differentiated or moderately differentiated,
nonfunctioning, somatostatin receptor-positive neuroendocrine tumors of grade
1 or 2 (a tumor proliferation index [on staining for the Ki-67 antigen] of
<10%) and documented disease-progression status. The tumors originated in the
pancreas, midgut, or hindgut or were of unknown origin. This is the CLARINET
study. However, NETest is not mentioned by any of the authors in this paper.
It is not supportive of NETest.

Strosberg et al. (67) This paper was reviewed by this contractor in a prior
reconsideration. It is also an excellent paper in the prestigious _New England
Journal of Medicine_ concerning use of 177Lu-Dotatate in patients with
advanced, progressive, somatostatin-receptor-positive midgut neuroendocrine
tumors. However, NETest is not mentioned by any of the authors in this paper.
It is not supportive of NETest.

De Wever et al. (68) This paper was reviewed by this contractor in a prior
reconsideration. NETest is not mentioned by any of the authors in this paper.
It is not supportive of NETest.

Allingham-Hawkins et al. (69) This paper was reviewed by this contractor in a
prior reconsideration. NETest is not mentioned by any of the authors in this
paper. It is not supportive of NETest.

NCCN Breast Cancer Version 2.2017 (70). This paper was reviewed by this
contractor in a prior reconsideration. However, NETest is not mentioned. It is
not supportive of NETest.

Smith et al. (71) This paper was reviewed by this contractor in a prior
reconsideration. Neither neuroendocrine tumor screening nor the NETest are
mentioned by any of the authors in this paper. It is not supportive of NETest.

Knigge et al. (72) This is another international (European Neuroendocrine
Tumor Society) consensus guideline on the standards of care. This article
discusses the follow-up and documentation of patients with neuroendocrine
tumors. It lists a number of tumor markers and specifically starts off with
Chromogranin A stating, "At present, the most common and reliable tumor marker
is plasma CgA for patients with G1 and G2 NEN [neuroendocrine neoplasms]." It
then mentions Chromogranin B, Neuron-Specific Enolase, 5-Hydroxyindoleacetic
Acid, Serotonin, Gastrointestinal Hormones (insulinoma, gastrinoma,
glucagonoma, VIPoma, somatostatinoma), and NT-pro-Brain Natriuretic Peptide.
However, it does not mention NETesting. In addition, it notes "The follow-up
should be in accordance with the ENETS consensus guidelines from 2011 and
2016, the present and coming WHO classification and ENETS/UICC recommendations
for TNM staging...However, it should be stressed that evidence-based studies for
follow-up are largely missing." The article concluded with "Disclosure
Statement: The authors have no conflicts of interest." This paper is not
supportive of NETest.

Atri et al. (74) This is another paper in the _New England Journal of
Medicine_. However, it describes how the consultant revaluated a patient who
turned out to have a neuroendocrine tumor. As the publication states, "In this
_Journal_ feature, information about a real patient is presented in stages
(boldface type) to an expert clinician, who responds to the information,
sharing his or her reasoning with the reader (regular type).The authors'
commentary follows." The author noted, "When a neuro endocrine tumor is
suspected as a cause of chronic diarrhea, the serum gastrin, vasoactive
intestinal peptide, and chromogranin A and urine 5-hydroxyindoleacetic acid
levels should be measured." NETesting is not mentioned, nor are any articles
by Dr. Kidd or Dr. Modlin referenced. This paper is not supportive of NETest.

Pallais et al. (75) This is another paper in the _New England Journal of
Medicine_. It is a "Case Records of the Massachusetts General Hospital" and
describes how an expert clinician evaluated a "45-year-old woman [who] was
admitted to this hospital because of dyspnea on exertion, fatigue, and
confusion" who turned out to have a neuroendocrine tumor. Chromogranin A is
mentioned as a neuroendocrine marker, but NETesting is not mentioned. The
third author, Dr. Lu "reports receiving consulting fees from PQ Bypass and
grant support from Nvidia." There were no other reported conflicts by any of
the authors. There are no articles by Dr. Kidd or Dr. Modlin referenced. This
paper is not supportive of NETest.

The following submissions were received by NGS during the comment period. They
have been reviewed and added to the existing list of references. They are
numbered as a continuation of the original reference list in this LCD.

Chen et al. (76) This is a very interesting research overview dealing at the
molecular level of cancers. The authors, "On the basis of genetic expression
data, classified 10,224 cancers, representing 32 determined major types, into
10 molecular based 'classes.'" The authors do not mention NETest by name, but
describe it without comment. The authors conclude their review with "there
being a clear need for additional and better biomarkers. The more accurate
identification of neuroendocrine-associated cancers in particular would have
important implications regarding the treatment of this disease." Disclosure of
Potential Conflicts of Interest. No potential conflicts of interest were
disclosed." This paper does not support, nor refute NETest.

Liu et al. (77) This study is based on NCT022705667 which is a registry on the
National Clinical Trials Database. This registry is estimated to be completed
March 2020. This paper was critiqued by Rindi and Wiedenmann (#175 above).
Neuroendocrine Neoplasia Goes Molecular--Time for a Change. 2018. Clinical
Oncology. https://doi.org/10.1038/s441571-018-0118-8. "In this study, 100
patients were monitored for 6-12 months.. Patients received the test at
enrolment, were assigned to two groups depending on whether they had stable
disease ('watch-and-wait' group) or progressive disease ('intervention' group)
according to Response Evaluation Criteria In Solid Tumors (RECIST) criteria,
and separately analyzed.

"Seventy-two patients received a single test, and the rest received 2-4 tests.
The data provided suggest tumour progression can be predicted earlier using
NETest than with established RECIST criteria" However, Rindi and Wiedenmann
noted, "The presented data, however, must be considered with caution, given
the focus of the study on neuroendocrine cancer. Neuroendocrine neoplasia
encompasses distinct tumour entities with growth behaviors that vary from
extremely slow to very rapid. The incidence and aggressiveness of
neuroendocrine neoplasms is highly dependent on the anatomical site of origin.
The most aggressive disease is frequently found in the pulmonary tract (in the
form of small-cell lung cancer [SCLC], and is also referred as neuroendocrine
carcinoma (NEC) when found at other anatomical sites. The most indolent cases
are frequently detected in the digestive system were previously termed
carcinoids and are currently referred to as neuroendocrine tumours (NETs).The
introduction of standard grading and staging tools for cancer in routine
practice in the past decade greatly improved our knowledge of neuroendocrine
neoplasia. Necessarily, inclusion criteria (according to anatomical site, and
staging and grading parameters) need to be accurately defined before
conducting studies in patients with neuroendocrine cancer in order to achieve
a clear understanding of the results. Liu and co-workers focused their
attention on patients with NET (97 of 100 patients tested). The most
aggressive poorly differentiated forms of neuroendocrine cancer were also
represent, but only in a very small subgroup (1 patient had SCLC and 2
patients had poorly differentiate NECs). The investigators used NETest to
evaluate blood samples of patients who predominantly had advanced-stage
neuroendocrine cancer (96 patients had stage IV disease), but with variable
disease grades, mostly, low grade (45 of 86 patients) or intermediate grade
(37 of 86 patients), and originating from different organs (68% in the
digestive tract and pancreas)....The emerging picture in this study of 100
patients is promising, but not all the results aligned with the expectation.
Discordance emerged when the NETest did not correlate with either absence or
presence of the disease (4%of patients) or with stable or progressive disease
status (17%and 23% of patients respectively) determined with stand methods. In
36% to 38% of patients in both groups the use of the test led to changes in
patient management, resulting in a reduction of the number of imaging
procedures performed.

"Unfortunately, the specificity and sensitivity of the test were not provided
nor could they be accurately calculated with the data made available; however,
the overall concordance with disease status reported (85) was well below 100%,
a value necessary to ensure that not a single patient is misdiagnosed and
therefore denied the most appropriate treatment. Understandably, reaching
perfection is almost impossible in the real word, and this principle holds
true especially in the complex management of patients with neuroendocrine
neoplasia. Nevertheless, a clear definition of the sensitivity and specificity
of NET is needed before the assay is widely adopted in clinical practice.
Furthermore, whether the test performs identically regardless of tumour grade,
tumour volume and/or treatment history remains unclear--the presented patient
numbers are too small and the selected cohort was to heterogeneous...Thus, the
cost-benefit radio needs to be accurately established once NETest has been
studied in larger and homogenous populations of patients with neuroendocrine
cancer" [note: Medicare does not use cost as a decision for coverage].
Disclosures. Scott Paulson: Ipsen, Taiho, Merrimack Advanced Accelerator
Applications, Bristol-Myers Squibb. The other authors indicated no financial
relationships. This paper does not support that NETest is proven to have
improved overall survival or is proven technology today. It is however a
promising paper.

Kulke et al. (78) This old paper deals with the important topic to "identify
key unmet needs, develop appropriate study end points, standardize clinical
trial inclusion criteria, and formulate priorities for future NET studies to
the US cooperative group program. Emphasis was placed on the development of
well-designed clinical trials with clearly defined efficacy criteria." The
authors do not mention NETest at all. They do propose that progression free
survival for both phase II and phase III studies be used rather than overall
survival. This view on endpoints is not standard today. Progression-free
survival is used to evaluate new drugs for cancers. The NETest is not a drug,
but rather a predictive test. This paper does not give any support to NETest.
Under Authors Disclosures of Potential Conflicts of Interest, a number of
honoraria and research funding related to drug companies were noted.

Singh et al. (79) This paper does not mention NETest. The introduction of the
article states, "Overall survival (OS) remains the gold standard end point for
randomized controlled clinical trials in patients with cancer." It states that
"[Neuroendocrine tumors] represent a tumor type in which [overall survival is
particularly difficult to evaluate because disease heterogeneity (resulting in
difficult patient recruitment in large Phase II trials), low numbers of
eligible patients (ie, those with proper diagnoses), and variable survival
times for patients with distal or regional [neuroendocrine tumors] (median OS,
33 or 111 months respectively)." Again, this is not a valid concern here. None
of the submitted NETest literature deals with Phase II or Phase III drug
trials. This is a paper from a drug company that wants to change the fact that
Overall survival (OS) remains the gold standard end point for randomized
controlled clinical trials in patients with cancer." The issue of using
overall survival (OS) rather than time to disease progression (TDP) or time
tumor progression (TTP) for phase II or phase III clinical trials is a
different is issue. Disclosure: Dr. Singh received research grants from
Novartis and consulting fees and honoraria from Novartis and Pfizer. Dr. Wang
is an employee of and a stockholder in Novartis Oncology. Dr. Law has received
research grants from Novartis Oncology and consulting fees from Pfizer
Oncology and Novartis Oncology and serves on the speaker bureau for Novartis
Oncology. This paper does not support NETest.

Ter-Minassian et al. (80) This paper does not mention NETest. It reflects the
same issues for changing the current standard of measuring endpoints for drug
trials. That is not the issue here. Acknowledgments: This study was funded in
part with a grant from Ipsen Pharmaceuticals. This paper does not support
NETest.

Imaoka et al. (81) This paper does not mention NETest. This paper's first
sentence is "In oncology clinical trials, overall survival (OS) is considered
the gold standard outcome measure." It reflects the same issues for want to
change the current standard of measuring endpoints for drug trials. That is
not the issue here. It does state, "The results of the present analysis
indicate the [progression free survival] is significantly correlated with
[overall survival], and suggest that progression free survival is an
acceptable surrogate for [overall survival] in clinical trials for
neuroendocrine tumors." Declaration of interest. "M I has received research
funding from Novartis Pharma K.K. and honoraria from Novartis Pharma K.K. and
is a member of advisory board for Novartis Pharm K.K., Teijin Pharma and Nobel
Pharma. The other authors have no conflicts of interest. This paper does not
support NETest.

Imaoka et al. (82) This paper does not mention NETest. It states, "In oncology
phase II trials, [overall survival] is considered the gold stand and is the
most commonly used primary endpoint....On the other hand, the primary goal in a
phase II trial to determine if there is sufficient evidence of antitumor
activity to undertake a phase III trial." The issue of NETesting in any
clinical trial of cancer drugs is not the issue. Disclosures: Masafumi Ikeda:
Novartis Pharma K.K. (RF, H), Novartis Pharma K.K., Teijin Pharma, Nobel
Pharma (SAB. The other authors indicated no financial relationships. This
paper does not support NETest.

NCCN Guidelines Version 3.2018--September 11, 2018 (83) The title page for
this National Comprehensive Cancer Network (NCCN) Guideline was submitted,
although with a page with the list of authors, and the Table of Contents.
Nothing else was included. However, NGS went to the NCCN site and looked up
the entire publication. There was not support for the NETest. This publication
does not support NETest.

Modlin et al. (84) This old article does not mention NETest. It does dwell on
improving the management of neuroendocrine tumors. There is no mention of
conflict of interest. This paper does not support NETest.

Oberg et al. (85) This paper has been reviewed previously and is #24 in the
LCD. This does not give any new support for NETest.

Oberg et al. (86) This paper has been reviewed previously and is #12 in the
LCD. This does not give any new support for NETest.

Dattani et al. (87) This paper does not mention neuroendocrine tumors or
NETest. It discussed the evaluation of "the oncological and survival outcomes
of a Watch and Wait policy in rectal cancer after a clinical complete response
(cCR) following neoadjuvant chemoradiotherapy." The tumors do not appear to be
neuroendocrine tumors. This paper does not support NETest.

Mazza et al. (88) While neuroendocrine tumors can occur about anywhere in the
human body, bladder neuroendocrine tumors must be very rare since no paper
submitted has mentioned any. There is no mention of any of these bladder
cancers being neuroendocrine tumors. This paper does not support NETest.

Bye et al. (89) This paper does not discuss NETest or neuroendocrine tumors.
It does not support NETest.

Moore et al. (90) This paper does not discuss NETest or neuroendocrine tumors.
It does not support NETest.

Odogwu et al. (91) This paper does not discuss NETest. It is concerned with
the use of Osimertinib. It does not support NETest.

Pavel et al. (92) This paper has been reviewed previously and is #35 in the
LCD. This does not give any new support for NETest.

Drukker et al. (93) This paper describes a proprietary gene prognosis method
on breast cancer. The proprietary gene prognosis method in this study is not
NETest. The breast cancers are not neuroendocrine tumors. It does not support
NETest.

Cardoso et al. (94) This paper in the prestigious New England Journal of
Medicine further describes the use of a 70-gene signature proprietary test on
women with breast cancer. However, the proprietary gene prognosis method in
this study is not NETest. The breast cancers are not neuroendocrine tumors. It
does not support NETest. Of note, none of the multiple articles submitted in
support of NETesting are from the New England Journal of Medicine although
this journal has published neuroendocrine tumor articles, none of which
mention NETest.

Burns et al. (95) This paper is from a plastic surgery journal. It does not
mention NETest. It does not mention neuroendocrine tumors. We agree with their
statement, "Physician are encourage to find the highest level of evidence to
answer clinical questions." This supports the obtaining the highest level of
evidence and does not support NETest.

Kidd et al. (96) This paper has been reviewed previously and is #37 in the
LCD. This does not give any new support for NETest.

Filosso et al. (97) This paper has been reviewed previously and is #38 in the
LCD. This does not give any new support for NETest.

Bodei et al. (98) This paper has been reviewed previously and is #33 in the
LCD. This does not give any new support for NETest.

Kidd et al. (99) This paper has been reviewed previously and is #21 in the
LCD. This does not give any new support for NETest.

Strosberg et al. (100) This paper has been reviewed previously and is #67 in
the LCD. This does not give any new support for NETest. Again, this is from
the New England Journal of Medicine which does publish neuroendocrine articles
but none of them as of yet has mentioned NETest.

Yao et al. (101) This paper in the prestigious New England Journal of Medicine
evaluates the new drug everolimus for the treatment of advance pancreatic
neuroendocrine tumors. Unfortunately, the article does not mention NETest at
all. Thus, it give no support to the use of NETest, especially for pancreatic
neuroendocrine tumors.

Kulke et al. (102) This recent paper evaluated everolimus combination with
Pasireotide LAR and everolimus by itself in advanced, well-differentiated,
progressive pancreatic neuroendocrine tumors. NETest was not used as a
biomarker for this study, although chromogranin A was. There is no mention at
all of NETest. This article was to evaluate the above cancer drugs. The
funding was by Novartis Pharmaceutical Corporation. Under disclosures there
were multiple concerning apparent conflicts of interest. This paper does not
support NETest.

Yao et al. (103) This paper evaluated everolimus in advanced, nonfunctional
neuroendocrine tumors of the lung or gastrointestinal tract. It is a drug
study. NETest was not used as a biomarker for this study. There is no mention
at all of NETest. The study was sponsored by Novartis Pharmaceutical
Corporation. This paper does not support NETest.

Caplin et al. (104) This paper has been reviewed previously and is #66 in the
LCD. This does not give any new support for NETest. Again, this is from the
New England Journal of Medicine which does publish neuroendocrine articles but
none of them as of yet has mentioned NETest. This does not support NETest.

Raymond et al. (105) This paper evaluated sunitinib malate for the treatment
of pancreatic neuroendocrine tumors. It is a drug study. There is no mention
of NETest. This does not support NETest.

Andersson et al. (106) This is a paper on imaging for appendicitis. This does
not pertain to neuroendocrine tumors. It does not mention NETest. This paper
does not support NETest

Modlin et al. (107) This paper has been reviewed previously and is #63 in the
LCD. This does not give any new support for NETest.

Modlin et al. (108) This paper gives a good overall view of neuroendocrine
tumors. However, despite the claim, NETest has not been independently
validated. The registry under NCT02270567 is mentioned. However, this
voluntary registry is still open and is not expected to be completed until
March 2020. There is no Conflict of Interest statement. This gives some
support to NETest.

Patel et al. (109) This paper gives a good overview of neuroendocrine tumors
including treatments It does specifically state, "Chromogranin A (CgA) is the
most established biomarker and is used for diagnosis and monitoring in
gastroenteropancreatic neuroendocrine tumors. It does give a brief discussion
of Nest citing only two articles. Based solely on two article and the Oberg et
al Consensus on Biomarkers for Neuroendocrine Tumour Disease (#12 in the LCD)
it concluded "Therefore NETest has a role in the identification of disease
progression, defining treatment, efficacy, and completeness of resection." NGS
considers the above Consensus opinion as limited support for NETest. The
Declaration of interest in the Patel paper shows N. Pavlakis as an advisor for
Amgen, Novartis, Pfizer and Roche Pharma; TJ Price on the advisory board for
Ipsen.

Capdevila et al. (110) This paper gives a good overall review of
neuroendocrine tumors. The authors acknowledge a grant from Novartis. It
devotes one paragraph which starts, "One of the main limitations of clinical
trials in patients with [neuroendocrine tumors] has been the lack of a potent
biomarker program to run alongside drug development." It then discussed NETest
citing "Blood and tissue neuroendocrine tumor gene cluster analysis correlate,
define hallmarks and predict disease status" in Endocr Relate Cancer. (Kidd M,
Drozdov I, Modlin I.)2015;22(4):561-575 which is reference #21 in the LCD. The
paragraph concludes with "Several ongoing clinical trials have included the
NETest validation and would probably substitute the classical monoanalyte
strategies for prognostic and predictive value of blood biomarkers in NETs
setting." This statement is not referenced. This paper gives weak support for
NETest.

Kyriakopoulos et al. (111) This review is a very baffling paper. It mentions
NETest twice. First it merely states, "NETest: The NETest is a multianalyte
algorithm analysis PCR-based test recently proposed using 51 'finger-print'
genes characterizing a NET. Its clinical value is under investigation and the
early reports are promising to predict treatment response as well as early
identification of recurrence." The only reference cited about this test is #34
in the policy (Cwikla JB, Bodei L, Kolasinska-Cwikla A, Sankowski A, Modlin
IM, Kidd M. Circulating Transcript Analysis (NETest) in GEP-NETs Treated With
Somatostatin Analogs Defines Therapy. J Clin Endocrinol Metab.
2015;100(11):E1437-E1445. DOI 10.121/jc.2015-2792) which studied only 35
patients for a median of 10 months. Then under Conclusions, it states, "Test
and programs such as the NETest and the MASTER have been already validated
from scientific groups and they have been proven effective to select patients
who will benefit from specific medications and to predict the response to
treatment." This contradictory statement is not supported in the article. This
publication does not offer robust support for NETest. It does not actually
mention any long term follow-up studies, improved overall survival or
independent, external validation of the results. This paper states, "The
authors have no conflicts of interest to declare."

Auernhammer et al. (112) This review article states, "We discuss clinical
development and controversies in the treatment of neuroendocrine tumours
(NETs) that are relevant for clinicians and clinical researchers." It notes,
"The serum tumor marker chromogranin A is used extensively as a biomarker for
tumour load but has limited sensitivity and accuracy. Improved biomarkers are
therefore urgently need. A novel blood-based multi-analyte transcript
analysis, encompassing 51mRNA analytes (NETest), had a much higher sensitivity
and specificity (90-98%) than chromogranin A. The readouts for NETest
correlated with NET tumor load, progressive disease, and response to
treatment, including surgery or cytoreduction, somatostatin analogues, or
peptide receptor radionuclide therapy (PRRT). Circulating tumour cells and
miRNAs are also novel prognostic blood-derived biomarkers for NETS (recently
reviewed by Zatelli and colleagues) but the utility of these biomarkers in
routine clinical practice has yet to be evaluated." The paper does not mention
NETest in any algorithm or treatment discussion. The "Summary and conclusion"
at the end of the article states, "The field of tumor biology and genetics of
small intestine and pancreatic NETs has grown substantially. Promising blood-
based biomarkers would be further evaluated in the real-world setting." We
agree with this need for further evaluations. Declaration of interests: CJA
received search contracts from Ipsen, Novartis, and ITM Solucin, lecture
honorarium from Ipsen, Novartis, and Falk Foundation, and advisory board
honorarium form Novartis. CS has received research grants from Novartis,
lecture honoraria from Ipsen and Novartis, and advisory board honoraria from
Ipsen, Novartis, and Pfizer. MKA has received research contracts from
Novartis. AG has received advisory board and lecture fees from Novartis and
Ipsen. SN has received research contracts from Novartis and lecture fees from
Ipsen. HI has received a research contract from Novartis, lecture honorarium
from Siemens, and advisory board honorarium from Bayer. PB has received a
research contract from Roche and advisory board honorarium from Advance
Accelerator Applications. SB, TK, JM, and MR declare to competing interests.
Also, they do not comment on improved clinical outcome on patients with
neuroendocrine tumors. This paper does not give support to NETest.

Ma et al. (113) This is an old historical paper explaining the authors'
attempts at developing microarray technologies into cancer research. This
paper does not mention NETest at all. No clinical use was described. This
paper merely shows their research and their vision for such testing in the
future. There were no disclaimers. This paper does not support NETest.

Erlander et al. (114) This is another historical paper explaining the authors'
attempts at developing microarray technologies into cancer research, but
solely in determining tumor classification. This paper does not mention NETest
at all. No clinical use was described. This paper merely shows their research
in trying to determine the origin of cancers. Disclaimer. The study was funded
by and completed at bioTheranostics, Inc. The authors are employees or former
employees and stockholders in bioTheranostics, Inc." This paper does not
support NETest.

Greco et al. (115) This paper describes determining the origin of metastatic
cancer in 38 out of 501 patients with cancer of unknown primary site (thus
7.6%). This used the technology of #39 above, and not NETest. The purpose was
to help identify the primary cancer site. The authors concluded, "This study
is too small to make any other meaningful correlations or conclusions
regarding the efficacy of therapy." Conflict of interest. This was funded by
bioTheranostics and Dr. Greco, Erlander, and Dr. Hainsworth list their
financial ties with bioTheranostics, Inc. This paper does not support NETest.

Den et al. (116) This paper describes using the Decipher genomic classifier
by GenomeDx Biosciences which is a whole transcriptome microarray as a
predictor for clinical metastases after radical prostatectomy with subsequent
radiation therapy. The test was not NETest. Prostate cancer is not a
neuroendocrine tumor. They concluded that patients with lower genomic
classifier scores may benefit from delayed radiation therapy as opposed to
those with higher genomic classifier scores. They cautioned "However, this
needs prospective validation." Conflicts of interest: Funding was provided by
GenomeDx and M.G., K.Y., and E.D. are employees of GenomeDx." This paper on
prostate cancer does not support NETest.

Den et al. (117) This is a follow up paper on #41 above. They concluded that
using genomic classifier scores was prognostic for the development of clinical
metastasis beyond routine clinical and pathologic features. The authors noted
that the data was analyzed retrospective and that the section of adjuvant
radiotherapy as oppose to salvage radiotherapy varied among physicians and
patients. They also not there were no concrete guidelines for the
incorporation of androgen-deprivation therapy with postprostatectomy
radiotherapy. Most important, this is not a paper on neuroendocrine tumors,
but on prostate cancer. Also, the testing was not NETest. Disclosures: There
were conflicts of interest noted and there was funding by Conflicts of
interest: Funding was provided by GenomeDx and M.G., K.Y., and E.D. are
employees of GenomeDx. This paper on prostate cancer does not support NETest.

Modlin et al. (118) This paper has been reviewed previously and is #64 in the
LCD. This does not give any new support for NETest.

NCCN (119) This paper has been reviewed previously and is #5 in the LCD. It
was also reviewed before that. This still does not give any support for
NETest.

Kulke et al. (120) Only three pages of this old article were sent. NETest was
not mentioned. The entire NCCN Neuroendocrine Tumors Vison 1.2015 was
subsequently obtained by NGS. A word search failed to reveal NETest. This
paper does not give any support for NETest.

Bowden et al. (121) This paper has been reviewed previously and is #10 in the
LCD. This does not give any new support for NETest.

Grosse et al. (122) This historical paper is on philosophical issues including
societal one and cost-effectiveness of genetic testing. It is not clinical. It
does not mention neuroendocrine tumors. Of note, Medicare is not allowed to
use cost as a reason for coverage. It does not mention NETest. This does not
support NETest.

Buyse et al. (123) This is a historical paper that argues that overall
survival on drug trials should be replaced by progression free survival for
advanced colorectal cancer. It is not about neuroendocrine tumors. Multiple
papers submitted from a later date show that overall survival has been
considered the "gold standard." In addition, see the related response by
Yothers below in reference 124 who notes, "Time to event can be ascertained
with excellent reliability for OS, but the difficulties in ascertaining time
to progression lead to less reliability for PFS." The "Authors' Disclosures of
Potential conflicts of Interest" note compensation from AstraZeneca to KC and
PP as well this paper coming from the "International Drug Development
Institute." This does not support NETest.

Yothers (124). This is an editorial about the Buyse study also in the same
journal. It does speak positively about Progression Free Survival but notes
that the Buyse article is about advanced colorectal cancer. It notes, "Time to
event can be ascertained with excellent reliability for OS, but the
difficulties in ascertaining time to progression lead to less reliability for
PFS." This paper comments on the Buyse being a drug trial, which as noted
below is different issue. The author indicated no potential conflicts of
interest. This paper does not support NETest.

Tang et al. (125) This is another historical paper that argues that overall
survival on drug trials should be replaced by progression free survival for
metastatic colorectal cancer. It is not about neuroendocrine tumors. Multiple
papers submitted from a later date show that overall survival has been
considered the "gold standard." It argues that "A validated shorter term
surrogate end point would reduce drug development costs and allow for more
rapid completion of randomized controlled trials." No conflicts of interest
were reported. Saving pharmaceutical companies money is not something that is
appropriate to use for LCD determination criteria. In addition, see the
related response by Montagnani et al below in reference 126. Again, this paper
does not deal with NETesting or neuroendocrine tumors. It does not support
NETest.

Montagnani et al. (126) In the same journal in response to the above Tang
article noted, TSurrogate end points of survival have been hypothesized (sic)
and validated for patients with metastatic colorectal cancer treated with
first-line FU-based cytotoxic chemotherapy. Outside this specific situation,
their utility is not clear. In particular, they could be useless when
considering newer drugs like BV and other antiangiogenic drugs. In conclusion,
the correlation between PFS and OS and between PFS and OS for patients with
metastatic colorectal cancer treated with BV-based bio-chemotherapy was not
demonstrated. Our opinion is that using BV combined with chemotherapy in
first-line treatments for patients with metastatic colorectal cancer survival
should remain the primary end point until validity of surrogate end points
will be clearly demonstrated." The authors indicated no potential conflicts of
interest. This paper does not support that progression free survival can be
used rather than overall survival for NETesting.

Heng et al. (127) This paper notes that overall survival has been the
traditional gold standard for all endpoints. This study deals with metastatic
renal cancers, not neuroendocrine tumors. They concluded that progression-free
survival at 3 months and at 6 months predicted overall survival on these renal
cancer patients and the current results indicated that profession-free
survival may be a meaningful intermediate endpoint for overall survival in
patients with metastatic renal cell carcinoma who receive treatment with novel
agents. They did conclude, "These results require prospective evaluation." As
noted below by Escudier,"There are several weakness this study some of which
are highlighted by the authors themselves. First, only 70% of the patients
received targeted agents as first-line treatment and therefore were really
eligible for the analysis performed. Second, PFS was not independently
reviewed and outside of clinical studies, which was the case for the majority
of the patients, the treating clinicians are often biased and may overestimate
PFS in patients with slow-growing disease. Consequently, such patients may
have a longer than expected OS. Finally, only 35% of the patients in the study
by Heng et al received additional (i.e., subsequent) therapy, which is
different from the current real-life situation, now that so many drugs have
been approved." Again, they evaluated metastatic renal cancer, not
neuroendocrine tumors. In addition, it seems like almost all the papers
submitted which NETest has used were a heterogeneous collection of
neuroendocrine tumors with multiple different organs of origin and different
degrees of spread. This paper does not support NETest.

Escudier (128) in the same issue of Cancer wrote a response editorial to the
above Heng study. "There are several weakness to the study by Heng et al, some
of which are highlighted by the authors themselves. First, only 70% of the
patients received targeted agents as first-line treatment and therefore were
really eligible for the analysis performed. Second, PFS was not independently
reviewed and outside of clinical studies, which was the case for the majority
of the patients, the treating clinicians are often biased and may overestimate
PFS in patients with slow-growing disease. Consequently, such patients may
have a longer than expected OS. Finally, only 35% of the patients in the study
by Heng et al received additional (i.e., subsequent) therapy, which is
different from the current real-life situation, now that so many drugs have
been approved. Conflict of interest disclosures: The author made no
disclosures." This paper does not support that progression free survival can
be used rather than overall survival for NETesting.

Modlin et al. (129) This paper has been reviewed previously and is #26 in the
LCD. This does not give any new support for NETest.

Cwikla et al. (130) This paper has been reviewed previously and is #34 in the
LCD. This does not give any new support for NETest.

Malczewska et al. (131) This is a single case report of a 52 year old woman
with a large ulcerated friable mass obstructing the ileocecal valve which was
a 5cm well-differentiated neuroendocrine tumor which was treated by a right
hemicolectomy and lymphadenectomy. Post-operatively her NETest was elevated.
Following 177Lu peptide receptor radionuclide therapy, the NETest measurements
were lower. Then it was elevated and the patient needed a cholecystectomy for
symptomatic cholelithiasis. The abdomen was normal but random multiple
peritoneal and core needle biopsies from the segment VI were performed. This
reveal foci of metastatic tumor but the tumor was too small for accurate Ki-67
assessment. No histological pathology report is mentioned. Somatostatin
analogues were increased. The NETest levels remained elevated but lower. The
authors concluded "A prospective clinical trial that included image-negative
but NETest-positive patients would be of value to demonstrate the clinical
utility of the liquid biopsy." We concur with this conclusion about a
prospective clinical trial. Disclosure summary. "AM, LB: Nothing to disclose;
MK: Laboratory Director of Wren Laboratories; IMM: Medical/Scientific
Consultant to Clifton Life Sciences." This single case report gives some
support to NETest.

Kaltsas et al. (132) This paper has been reviewed previously and is #54 in the
LCD. This does not give any new support for NETest.

Bodei et al. (133) This paper has been reviewed previously and is #11 in the
LCD. This does not give any new support for NETest.

Genc et al. (134) This paper listed as "RESEARCH ARTICLE" 35 patients with
pancreatic neuroendocrine tumors. Originally there were 124 patients operated
on for pancreatic neuroendocrine tumors at Academic Medical Center Amsterdam
between 2006 and 2015. 89 were excluded: 21 were deceived; 6 were R2 or
unknown resection margins; 6 withdrew consents; and 56 were lost to follow-up.
Completely excised tumors were classified as R0 and tumors with microscopic
involvement <1 mm were classified as R1. R2 was not defined and there are
several classifications of resection margins, but most likely the R2
designation for macroscopic residual tumor. The three groups were R0
(Completely excised tumors) with no recurrence which had n=11; R0 (Completely
excised tumors) with recurrence (n=12) and R1 (tumors with microscopic
involvement <1 mm) with no recurrence (n=12). The recurrence of the second
group (R0 (Completely excised tumors) with recurrence) had 3/12 (25%) being
local recurrence, 2/12 (17%) being regional recurrence, and 7/12 (58%) being
distant. Of further note, 73% of the first group where Grade I tumors and 27%
Grade II, while the second group had 42% Grade I and 58% Grade II, but the
third group was 100% grade I. The results are measured in median, not mean.
However the median follow up for group 1 was 31 months, for group 2 105
months, and group 3 92.5 months. NETest levels were higher in group 2 than in
group 1 or group 3. NETest gave false positive or negative re in 18% using a
40% cutoff. The authors concluded "Larger prospective studies are warranted to
more fully explore the utility of the NETest in the identification of
postoperative residual pancreatic NET disease or recurrence and to help better
stratify patients for post-surgery treatment. This study is interesting. If
suffers from several things. Most of the potential enrollees (89 out of 124)
were excluded. The study was really retrospective since it included patients
as far back as 2016. There does not seem to be any clear evidence that the
results of the NETest was used to treat the patients. There is no evidence
that the NETest improved overall survival. We agree that this gives some
support for NETest and will welcome larger prospective studies to more fully
explore the utility of the NETest especially on overall survival. Disclosure
and Funding information: "Funding information: Ipsen Fund." IM Modlin is a
Medical/Scientific Consultant to Wren Laboratories. M Kidd is the Laboratory
Director of Wren Laboratories. I Drozdov is a Bering, Statistical consultant
to Wren Laboratories."

Ward et al. (135) This is a review of gene expressions and the treatment of
breast cancer. It only discusses six tests, and none of them are NETest. It
also considers cost which Medicare cannot use. This paper does not support
NETest.

Sparano et al. (136) This paper in the prestigious New England Journal of
Medicine does not support NETest. Actually it in a way hurts it since it shows
that other gene-expression assays get published in this journal, but NETest
results are not. This paper deals only with breast cancer and a different
gene-expression assay. It has nothing to do with neuroendocrine tumors, but
only breast cancer. Conflicts of interest are not noted. This does not support
NETest.

Krop et al. (137) This is another paper giving guidance to physicians treating
breast cancer. It is based on the use of genetic tests, but not those of
NETest. These are not neuroendocrine tumors. There is no mention of NETest.
Authors' Disclosures of Potential Conflicts of Interest. Ian Krop: employment,
leadership and stock/other ownership AMAG Pharmaceutical. Consulting or
advisory role: Genentech. Research Funding: Genentech; Other Relationship:
Novartis. Nofisat Ismaila. No relationship. Fabrice Andre: Research funding
AstraZeneca, Novartis, Pfizer, Eli Lilly. Travel, Accommodations, Expenses:
Novartis, Roche, GlaxoSmithKline, AstraZeneca. Robert Best: Research funding
Arrien; Patents, Royalties, Other Intellectually Property: Fujirebio
Diagnostic's royalties for CA0125 ovarian cancer biomarker. Travel,
Accommodations, Expenses: Roche. William Barlow: Research Funding:
AstraZeneca, Merck. Deborah Collyar: No relations. M. Elizabeth Hammond: No
relationship to disclose. Nicole Kuderer: Consulting or Advisory Role:
Jannsen, Coherus Biosciences, Halozyme, G1 Therapeutics, Myriad Genetics.
Research funding Amgen, Travel, Accommodations, and Expenses: Janssen, coheres
BioSciences. Minetta Liu: Research Funding Eisai, Seattle Genetics, Celgene
Veridex, Novartis, Genentech, GRAIL Merck. Travel, Accommodations, Expenses:
GRAIL, Merck, Celgene. Robert Mennel: Employment: Texas Oncology. Catherine
Van Poznak: Research Funding Bayer; Patents, Royalties, other intellectual
Property: UpToDate. Antonio Wolff: Consulting or Advisory role Ionis. Research
funding: Myriad Genetics, Pfizer. Patents, royalties, other intellectual
property: Named inventor on one or more issued patents or pending patent
applications relating to methylation in breast cancer with rights assigned to
Johns Hopkins University and participation in a royalty-sharing agreement with
Johns Hopkins. Vered Stearns: Research Funding: Abbvie, Merck, Pfizer,
MedImmune, Novartis, Celgene, Puma Biotechnology. This paper does not support
NETest.

NCCN (138) This is another paper giving guidance to physicians treating breast
cancer. It is based on the use of genetic tests, but not those of NETest.
Breast cancers are not neuroendocrine tumors. There is no mention of NETest in
this guideline. This paper does not support NETest.

Cives et al. (139) This is an overall review of epigenetic modifications
(phenotypic trait variations by external or environmental factors that switch
genes on and off and affect how cells respond to genes instead of being caused
by changes in the DNA sequence) in cancers. The paper does not mention NETest
by name but describes it, noting, "A multianalyte whole blood RNA multigene
signature has been developed to predict NET activity. Interesting, among the
omic clusters capable of differentiating disease activity (sable versus
progressive disease), there was the epigenome. Future studies should assess if
the epigenomic profiling per se may serve as a predictive biomarker for
treatment tailoring in NET patients." Conflicts of interest: The authors
declare no affiliation with industries or organization with a financial
interest, direct or indict, that may affect the conduct or reporting of the
work submitted." This paper does not support that NETest is standard.

Pawa et al. (140) This is a manuscript and there is no published information.
Upon checking PubMed Data Base of medical literature, from the US National
Library of Medicine, National Institutes of Health, it is noted that this
journal has not been included in their index of medical literature since March
2013. Unpublished papers are not adequate peer reviewed literature as indexed
in the required PubMed Data Base of medical literature, from the US National
Library of Medicine, National Institutes of Health. Thus this does not support
NETest.

Oberg et al. (141) This paper has been reviewed previously and is #58 in the
LCD. This does not give any new support for NETest. Of note, upon checking
PubMed Data Base of medical literature, from the US National Library of
Medicine, National Institutes of Health, it is noted that this journal has not
been included in their index of medical literature since March 2013.

Svensson J. (142) Targeted Radionuclide Therapy for Patients with
Neuroendocrine Tumours. 2016. Department of Oncology, Institute of Clinical
Sciences, University of Gothenburg. No other information is available about
this. Beside the above noted cover sheet, there one sheet labeled "13" on the
bottom with a paragraph stating, "A newer concept to evaluate respons [sic] to
treatment is to analyses [sic] changes in disease specific gene transcript in
blood. NETest is a PCR-based analysis of a 51 NET-associated genes, and
changes in the transcript profile is reported to have delineated surgical
cytoreduction {113] as well as treatment response from 177Lu-DOTATATE [114].
It was also observed that this test could predict response to somatostatin
analogue treatment using octreotide [115]." This poorly reference source does
not give acceptable support to NETest.

Verbeek et al. (143) This paper has been reviewed previously and is #31 in the
LCD. This does not give any new support for NETest.

Freis et al. (144) This paper reported on the various prognostic factor in
neuroendocrine carcinomas, using overall survival as the endpoints. While they
used a number of laboratory tests, they noted, "Recent studies have also
reported the potential of blood transcript analysis as a predictor and
prognostic marker of progression in well-differentiated NET, but this was not
studied in in NEC (neuroendocrine carcinoma patients. Moreover, molecular
analysis such as p53 and retinoblastoma (RB) protein staining are probably
promising in NEC, but these analyses are not validated for survival prognosis
in NEC." "Competing financial interests: The authors declare no competing
financial interests." This paper does not support NETest although it supports
the use of overall survival in determining results in neuroendocrine papers.

van Treijen et al. (145) This paper is a "systematic review [aimed] to
evaluate current literature and amplify an up-to-date evidence-based approach
to [nonfunctional pancreatic neuroendocrine tumors] diagnosis in [multiple
endocrine neoplasia type 1]" the authors state the electronic bibliography
databases Medline/PubMed, Embase, and Web of Science were searched December
2017 to review systematically current literature on the diagnostic value of
biomarkers and imaging modalities for [nonfunctional pancreatic neuroendocrine
tumors] in in [multiple endocrine neoplasia type 1] patients....To gain insight
into the penetranc3 and behavior of for [nonfunctional pancreatic
neuroendocrine tumors] in in [multiple endocrine neoplasia type 1 and
subsequently answer the question on the optimal timing and frequency of
follow-up, a third search was operated, also including our study domain in
[multiple endocrine neoplasia type 1] patients. The literature searches were
reviewed by an experience librarian. Database subject terms such as Mesh terms
(Medline) and Emtree terms (Embase), were used as appropriate. Selection of
articles was restricted to English, Dutch, German and French, and for original
research, there was no restriction for the year of publication of the
studies." It is confusing about the second search since there is not much
mention of it. Despite the massive effort to locate pertinent papers, the
authors did not evaluate NETest, apparently because they could not find
supportive literature. They did mention under "Discussion" that, "Future
studies should investigate the role of molecular biomarkers, such as the
NETest for [multiple endocrine neoplasia type 1-related [nonfunctional
pancreatic neuroendocrine tumors] as the current screening tools lack insight
in the dynamics of individual tumor behavior." "Disclosure Summary: The
authors have nothing to disclose." This paper does not support NETest.

Banck et al. (146) This is a review "summarizing progress in clinical trials
and basic science redefining the diagnosis and treatment of well
differentiated small intestine neuroendocrine tumors (SI-NET)." Just before
the CONCLUSSION, there is a section entitled "NOVEL DIAGNOSTIC TOOLS." It
states, "A blood test for tumor-derived RNA was reported by Modlin, et al. The
test uses reverse transcriptase polymerase chain reaction (RT-PCR) to quantify
51 NET marker-transcripts and control transcripts in patient whole blood. The
sensitivity and specificity of the test to determine whether a patient has a
NET was 79-88t and 94%, respectively, suggesting that it is superior to
measuring chromogranin A (specificity 85, sensitivity of 68%...How the test
score is computed from the 51 transcripts appears not to be fully documented
in the report. Furthermore, while blood samples from various clinical centers
were employed in the test development, additional studies such as prospective
measurements in clinical trial of NET treatment will be required to determine
the utility of the test for making clinical decision." This does not support
that NETest is standard and/or proven yet and does not support NETesting.

Maxwell et al. (147) This is another review paper on neuroendocrine tumors
(NETs). The paper makes a number of points. "The plasma serotonin assay is now
considered by reliable for the diagnosis of carcinoids when performed by CLIA-
licensed and College of American Pathology (CAP)-approved commercial
laboratories in the United States. This test a positive predictive value of
89% and negative predictive value of 93% of patients with midgut carcinoids,
but is less accurate in those with foregut and hindgut carcinoids. It may not
correlate as well with the tumor burden as other laboratory assays (e.g.
chromogranin, pancreastatin) because platelets become saturated at high level
of serotonin. It notes in the next paragraph, "Serum CgA (chromogranin A)
levels are considered one of the most useful markers for diagnosis and
surveillance of patients with gastrointestinal (GI) NETs, including hindgut
and foregut tumors, where 5-HIAA and serotonin levels are often with normal
limits." It also states, "The National Comprehensive Cancer Network (NCCN)
guidelines for GI NETs suggest testing for CgA and collecting a 24 hour urine
for 5-HIAA, but do not give specific recommendations for follow-up. It later
notes, "The NCNN recommends checking PP, CgA, calcitonin, PTH-rP, and GHRH for
generic PNETs. If the patient has a recognizable syndrome they recommend
checking specific hormone levels. The "WREN Assay" is discussed under
BIOMOLECULAR DIAGNOSITCS IN NETs. They note, "Modlin et al. set out to
identify a genetic signature for NETs that could be tested form peripheral
blood samples that might be useful for diagnosis, assessment of tumor burden,
and response to therapy. The authors concluded that their panel could identify
GEP NETs regardless of primary tumor site or metastasis, which could be useful
for screening and potentially response to therapy. The group is actively
recruiting patients to determine how well it might perform under these
circumstances." "Acknowledgments: Dr. Maxwell' work is supported by NIH
5T32#CA148062-05." This does not support that NETest is standard and/or proven
yet and does not support NETesting.

Bowden et al. (148) This paper was submitted twice by Wren Laboratories. This
paper has also been reviewed previously and is #10 in the LCD. This does not
give any new support for NETest.

Barbieri et al. (149) This is another review article about neuroendocrine
tumors. It states, "To date, the most widely used biomarker is plasma
chromogranin A (CgA), although its sensitivity and specificity in monitoring
therapy response is debated. Specific hormone products in functional NETs have
also been monitored. More recent, circulating tumor cells (CTCs), a panel of
neuroendocrine neoplasm gene signature for PCR-based blood analysis and MiRNA
profiling have been proposed as new potential prognostic markers. This is the
only reference to using NETesting in the article. There are no conflicts of
interested noted. This does not support that NETest is standard and/or proven
yet and does not support NETesting.

Chan et al. (150) The authors note, "This review was undertaken to evaluate
the literature on tissue-based as well as molecular imaging derived biomarkers
for neuroendocrine tumours, in order to identify areas where evince of
biomarker is robust and where gaps exist so as to direct further research." In
the introduction, it is noted, "Several reviews have focussed [sic] on blood-
based biomarkers in NETs. Serum-based assays offer advantages in terms of
convenience and safety of collection (compared to repeat tissue biopsies).
However, older studies have not demonstrated sufficient power to enable
accurate prognostication and have in general looked at individual measures in
isolation. Newer studies with multiple analyses show promise, but have not
been validated as yet." The paper discusses chromogranin A (CgA). It noted
that "Elevated baseline CgA has been shown to predict poorer overall survival
in multiple retrospective series...but other series fail to show this
correlation." It notes "Alterations in CgA with therapy may also be an
indicator of prognosis...this confirmed the prognostic but not predictive power
of CgA as a biomarker." It notes, "Unfortunately, there are several practical
issues associated with adoption of CgA as the only prognostic biomarker. False
elevations of CgA occur with benign conditions, other malignancies as well as
medication such as proton pump inhibitors, although the elevations is usually
mild... the reference range for CgA may also vary depending on the exact assay
utilized, hampering its interpretation and analysis across multiple clinical
centres. Elevated chromogranin A may simply reflect increased tumour
burden...and its independent contribution as a biomarker may decrease
particularly as improvements in imaging (with 68Ga and FDB PET scans) result
in improved staging of NETs. Finally CgA being a secretory product is produced
less in poorly-differentiated tumours, meaning that it is a poorly sensitive
marker for de-differentiated (poor prognosis) disease. Under "3.2.3 Other
plasma-based markets in NET" the author note, "A novel multi-transcript
molecular signature, the TET3st, is a blood-based assay examining a targeted
RNA expression profile. It has been investigated in 130 patients with GEPNETs...
with high sensitivity of 85098% and specificity of 93-97%. It showed accuracy
of 92-93T for NET when used to evaluate a mixed cohort of NET and non-NET
pathology specimens from the GI tract...This NETest score was also higher in
patients with clinically progressive disease compared to those with stable
disease...and subsets within the NETest score may reflect response to
somatostatin analogues...However, a prospective validation of these findings and
their prognostic significance at baseline is still ongoing." There are no
disclosers from the authors. This gives weak support of NETest.

Strinovic et al. (151) This is a review paper about neuroendocrine tumors with
minimal mention of NETest. It notes, "Recent studies investigate multiple
bowel NETs which have the potential to act as biomarkers but further studies
to define molecular mechanisms and validate these miRNA are needed. Andersson
et al. have defined specific gene expression patterns associated with tumor
grade and chromosomal alteration transcriptome of small bowel NETs as novel
prognostic biomarkers. Darmanis et al. proposed a new potential protein
biomarker for classify well-differentiated small intestinal NETs but further
investigations of these proteins in larger sample sets are needed. In
conclusion, chromogranin A and gastrin should be measured in all patients with
d-NETs. Serum NSE should be measured in all patients with d-NEC and G3 d-NETs
should. 5-HIAA should be measured only in patients with signs and symptoms of
carcinoid syndrome. Patients with serum gastrin>1000 ng/L should undergo ph-
metry and the status of parietal cell antibodies should be obtained. This is
important in order to classify the functional status od [sic] d-NETs.
Measurement of serum calcium parathyroid hormone, prolactin, IGF-1, ACTH,
urinary-free cortisol should be made in all patients as screening for MEN-1
syndrome." There is no mention of any role for NETest. Page 185 of this
article has a flowsheet on the workup for D-NET. There is no mention of NETest
although other standard biomarkers such as Serum CgA and gastrin are
specifically mentioned. There are no disclaimers. This; paper not only does
not support NETest, but denies there is any established role for it.

Xavier et al. (152) This is another review paper about neuroendocrine tumors.
Under BIOMARKERS AND OTHER LABORATORY TESTS, it states, "Several circulating
tumor markers have been evaluated for the diagnosis and follow-up of NETs.
Currently chromogranin A (CgA) is the most important of these markers, and
current guidelines recommend be measurement of serum CgA at diagnosis." In the
same section, it does note, "In an effort to find better NETs biomarkers,
Modlin e al recently published a multi-transcript molecular signature for PCR
blood analysis which may facilitate future diagnosis of NETs. They analyzed
transcripts of 3 microarray datasets (NETs peripheral blood, NETs tissue and
adenocarcinoma) and found 51 significantly elevated transcript markers. Based
on that, genetic based classifies were created and were able to detect NETs
with high sensitivity (85%-98%), specificity (93%-97%), positive predictive
value (95%-96%) and negative predictive value (87%-98%). The transcript marker
was similarly effect in recognizing pancreatic and gastrointestinal NETs, as
well as, metastases. Moreover, the gene-based classifier was significantly
more accurate than CgA and, in patients with low CgA, the transcript markers
were elevated in 91% of cases. This may reflect the future utility of genetic
makers as diagnostic tools of NETs, however further investigation needs to be
done to validate this hypothesis." There were no conflict of interest noted.
While this supports further investigation of NETest, it does not support that
it is currently proven to be of value and standard.

Jimenez et al. (153) This is not a valid article submission and thus does not
support NETest. Draft, unpublished literature and not appropriate to review.

Halfdanarson et al. (154) This is a review paper on biomarkers in
neuroendocrine tumors. It briefly discusses NETest, although not by name.
Under Novel tumor markers, the authors note, "A different approach is to
identify a molecular gene signature measurable in peripheral blood to detect
NETs. A multi-transcript molecular signature detected with a PCR analysis was
compared with CgA measurements in 176 samples of patients with malignancies,
including NETs. The gene-based classifier reliably detected patients with NETs
and was significantly more accurate than CgA for detecting Nets in patients
with low CgA levels. This promising approach to diagnosis of patients with
NETs awaits validation in larger studies." Also in Conclusions, the authors
note, CgA is currently the only circulating general NET marker in widespread
use and is both sensitive and specific for diagnosis of NETs, but nevertheless
suffers from significant limitations. ...Other markers should be used as
indicated based on the clinical presentation of the patients, especially in
cases of functional PNETs. Novel markers such as detection of CTCs and
molecular gene signatures hold great promise, but await further validation in
larger studies." There were no "Financial & competing interests disclosures"
listed for the authors. This paper does not support NETest being standard
today.

Chabot et al. (155) This is an editorial that states a very important tenet of
medicine and Medicare: Because of this variation in tumor biology, the dictum
'first, do not harm' should be a constant whisper in the surgeon's ear when
managing PNETs." It states, "The fourth article in this issue of Surgery by
Modlin et al describes the utilization of a 51-g3n expression panel from the
patient's blood to determine the efficacy of PNET treatment. Perhaps the
application of similar technology to blood and/or biopsies of PNETs could more
effectively discriminate those tumors that might be observed safely. The
article referenced is Modlin IM, Frilling A, Salem RR, et al. Blood
measurement of neuroendocrine gene transcripts defines the effectiveness of
operative resection and ablation strategies. Surgery. 2016;
159(1):336-347.This article has been reviewed at least twice before. Dr.
Chabot does not give any true support for NETest or reflect on whether or not
it is standard or effective. This editorial does not support NETest. We agree
with the author's premise "first, do not harm."

Manoharan et al. (156) This is another review article. The authors note,
"Previously, Modlin and coworkers introduced a new PCT-based blood-based
multianalyte neuroendocrine gene transcript assay (NETest) for the diagnosis
of gastroenteropancreatic neuroendocrine neoplasia (GEP-NEN)...The NETest
comprises 51 multigene transcripts...The results of this test were compared to
expression of chromogranin A in sporadic GEP-NEN. The NETest was more
sensitive compared to the common tumor marker. The sensitivity/specificity
rate was about 92.8%/92.8%...this NETest was than [sic] examined pre-and
postoperatively in 27 patients with GEP-NEN to monitor the therapeutic
affect...In 23 of 27 patients, the NETest result was significantly reduced
(preoperative: 80+/-5%, postoperative: 29+/-, P<0.0001 after curative
resection of the GEP-NEN. There are yet no data published regarding the value
of the NETest in MEN1 patients. However, if initial results can be confirmed
in large prospective MEN1 cohorts, the NETest might be a valuable screening
modality for MEN1 patients." The authors declared "There is no conflict of
interest that could be perceived as prejudicing the impartially of this
review." This paper does not support that NETest is standard today.

Basuroy et al. (157) This review article notes, "Circulating and tissue
markers of NET disease can be used to assess for disease prognosis, treatment
response, and recurrence. Existing markers may be general, like chromogranin A
(CgA) and serotonin, or specific to subtypes of NETs like gastrin and insulin.
More novel markers, like mRNA transcript panels and circulating tumor cells,
are likely to have a role in the future." There are no disclaimers." This
paper does not support that NETest is standard today.

Clift et al. (158) Only a manuscript like, unpublished format was submitted.
It is difficult to read because of its format. This is not acceptable as
evidence for support of NETest. Draft, unpublished literature and not
appropriate to review.

Barriuso et al. (159) This review article mentions NETest. "Promising results
have been presented over the last few years using a novel biomarker candidate,
the multianalyte algorithm analysis PCR-based test (NETest)." This test is
described. It then states, "In conclusion, this qPCR-based blood biomarker
test that defines the circulating fingerprint of a NET can be easily repeated
at multiple time points during periods between sequential imaging assessments,
thus providing additional real-time dynamic evidence regarding tumour behavior
as well as treatment efficacy. Confirmation of these observations in larger
series and randomized trials will allow the identification of likely non-
responders and have clinical utility in treatment decision making." Conflicts
of inter: JB has received research funding from Pfizer, Novartis and Ipsen. AC
has received fees from Pfizer, Ipsen and Novartis. JC has received fees from
Novartis, Ipsen and Pfizer. EG has received fess form Pfizer, Ipsen, Lexicon
and ADACAP, and Threshold Pharmaceuticals. MM has received honoraria as
speaker from Novartis and Ipsen. The other authors declared no conflicts of
interest. This paper shows potential for NETest but does not support that
NETest is standard today.

Hofland et al. (160) This review paper discusses many biomarkers, including
NETest which it describes and discusses. It noted, "Although these outcomes
certainly appear promising, there are concerns about the availability and cost
of this biomarker for the diagnosis of NETs in clinical practice. In addition,
the specificity of the NETest in population of patients with gastrointestinal
disease or other malignancies remains to be determined in more detail. Under
"Key Points" it noted, "Circulating transcript represent an emerging
opportunity in the diagnosis of gastroenteropancreatic NETs, but whether they
can be used to differentiate NETs form other tumours should be subject to
further study, while their availability and cost-effectiveness in clinical
practice remain to be confirmed. The paper concludes with "With the advent of
tests to measure circulating tumour cells, mRNA transcript and superior
functional imaging, our clinical repertoire is sure to notably expand and
positively affect patient care in the coming years. The authors declared no
competing interest. Note that Medicare does not use cost for determining any
coverage. This paper shows potential for NETest but does not support that
NETest is standard today.

Thorns et al. (161) This review article discusses microRNA testing. It notes
"...have been complemented by a first report on the establishment of a gene
expression-based classifier generated from peripheral blood samples from
patients with gastroenteropancreatic neuroendocrine tumors. While this
sentence does not mention NETest by name, it is describing NETest. It notes
that "Evaluation of microRNAs appear to be promising in the assessment of pNEN
[pancreatic neuroendocrine neoplasms]. In particular, miR-193b, which is also
increased in serum, may be a potential new biomarker of pNEN." miR-193b
however is not associated with the NETest description. Conflicts of interest
state CT and GB received funding from IPSEN Pharma GmbH, Novartis Pharma GmbH.
The other others had nothing to disclose. This paper does not support that
NETest is standard today.

Zatelli M. (162) This appears to be a teaching case report in a book
concerning a patient with a neuroendocrine tumor. It does note that "...multi-
analyte biomarkers have the potential for higher diagnostic sensitivity and
specificity as well as prognostic value ...Along the line of multi-analyte
strategies, multigene signatures have been considered as potential useful on
clinical grounds in order to provide real-time information about tumour
activity and response to treatment. In these setting, transcript analysis
provides copious information that has been employed to implement multi-analyte
assays with algorithm analyses (MAAA). The NET MAAA biomarker panel has
already been employed displaying high sensitivity and specificity, not
depending on age, gender, ethnicity, fasting or medications. However, this
method requires a dedicated laboratory and the necessary technical hardware,
software and skilled personnel that are not widely available." The case study
ends on "Up to Date of the Topic" by noting, "The issue of prognostic markers
in the NET field is highly debated and generates more reviews that [sic]
original research articles. " It then mentions mTOR pathways profiling might
have a prognostic role in lung and pNET. However, validation studies are still
lacking, both from the technical and clinical point of view." There are no
disclaimers. This short case study does not support that NETest is standard
today.

Lewis et al. (163) This brief review paper notes, "A novel 52-gene PCR-based
assay performed on peripheral blood may prove to have higher sensitivity and
specificity than the CgA biomaker [sic]." It further notes, "Because the assay
hinges on the circulating transcript signature of NETs, all 52 included genes
were validated as having mRNA that are reliably detectable in the peripheral
blood. Both at the time of diagnosis and when measuring responses to therapy,
this approach may permit more sensitive and specific test of GI NETs than CgA.
The general application of such an approach will require prospective
validation." There were no conflicts of interest. This short paper does not
support that NETest is standard today.

Ramachandran et al. (164) We fail to see any mention of NETest in this short
review paper. This short paper does not support that NETest is standard today.

Basuroy et al. (165) This brief review article devotes three sentences to the
summary of "Modlin IM, Drozdov I, Alaimo D, et al. A multianalyte PCR blood
test outperforms single analyte ELISAs (chromogranin A, pancreastatin,
neurokinin A) for neuroendocrine tumor detection. Endocr Relat Cancer.
2014;21(4):615-628" which is reference #29 in the current LCD. In "Practice
Points" IT NOTES, "Circulating gene transcript (mRNA) and tumor cells resent
potential NET biomarkers of interest that require further evaluation." It also
notes in that section, "Chromogranin A is used in routine clinical practice
and is associated with tumor burden, response and survival. Financial &
competing interests disclosure: J Ramage and D Sarker and note various
payments from Novartis and Pfizer and Dr. Ramage also had fees from Ipsen.
This paper does not support that NETest is standard today.

Zatelli et al. (166) This review paper describes the NETest. They note, "The
NETest appears to be a very promising tool not only for the diagnosis but also
for the follow-up of NET patients, although to date no information is
available concerning a predictive role for the NETest as concerns rapalog
sensitivity." Funding: M C Zatelli received consultant fees from Novartis and
Genzyme. This paper does not support that NETest is standard today.

Patel et al. (167) This review describes NETest and previous published
results. It concludes the paragraph with "Therefore, NETest has a role in the
identification off disease progression, defining treatment efficacy, and
assessment of completeness of resection." However, this statement is
attributed to "Oberg K, Modlin IM, De Herder W, et al. Consensus on biomarkers
for neuroendocrine tumour disease. Lancet Oncol. 2015;16(9):e435-e446"which is
reference #12 in the LCD. A concern about this endorsement was the fact that
this meeting was supported by Clifton Life Sciences, which owns Wren
Laboratories, and eight of the sixteen panelists are listed as having
financial ties to Wren Laboratories. Declaration of interest. N Pavlakis is an
advisor for Amgen, Novartis, Pfizer and Roche Pharma AD and TJ Price is on the
advisory board for Ipsen. Again, this gives the same limited endorsement for
NETest that reference #12 in the policy gives.

Razzore et al. (168) This short review paper does not mention NETest by name
but there is mention about it under "IS THERE A CLINICAL ROLE FOR NOVEL
BIOMARKERS? They note, "There is also increasing interest in miRNAs s clinical
biomarkers of tumorigenesis, treatment response and outcomes, but to date
clinical data are scarce and clinical applications challenging. Similarly,
there are several novel monoanalyte assays (i.e. connective tissue growth
factor for carcinoid heart disease (CCN2) or paraneoplastic Ma antigen 2
(NMA2) for small intestinal neuroendocrine tumors, but these analyses are not
available in clinical practice. Further, panelists of the recent Delphi
consensus gave the strongest support to the use of emerging biomarkers in
multianalyte based on genomics." Again, this statement is attributed to "Oberg
K, Modlin IM, De Herder W, et al. Consensus on biomarkers for neuroendocrine
tumour disease. Lancet Oncol. 2015; 16(9):e435-e446"which is reference #12 in
the LCD. A concern about this endorsement was the fact that this meeting was
supported by Clifton Life Sciences, which owns Wren Laboratories, and eight of
the sixteen panelists are listed as having financial ties to Wren
Laboratories. There was no financial support or conflicts of interest
reported. Again, this gives the same limited endorsement for NETest that
reference #12 in the policy gives.

There is a front sheet in a foreign language that we cannot read. (169) It is
followed by a page without any header or footer. This is not acceptable
medical support for NETest.

Capdevila et al. (170) This short review article notes, "We need pretreatment
biomarkers to be used as predictive factors of response, in order to improve
patient selection. In this line, the recent development of transcript blood
profiles in NETs (NETest) has demonstrated a higher sensibility than classical
biomarkers, such as chromogranin A and even tumor grade, and a better
predictive value of response to several treatment approaches in this field,
such as somatostatin analogs, peptide receptor radionucleotide therapy,
surgical resections and locoregional ablation procedures. The blood NET
transcript analyses also allow the monitoring of the disease evolution
favoring a real-time dynamic assessment and may have an impact in decision-
making process. Several ongoing clinical trials have included the NETest
validation and would probably substitute the classical monoanalyte strategies
for prognostic and predictive value of blood biomarkers in NETs setting. The
reference for the statement is from Kidd M, Drozdov I, Modlin I. Blood and
tissue neuroendocrine tumor gene cluster analysis correlate, define hallmarks
and predict disease status. Endocr Relat Cancer. 2015;22(4):561-575 which is
reference #21 in the LCD and previously had been reviewed. We noted that there
was no evidence of improved outcomes are noted. This has previously been
submitted and reviewed by this contractor in a prior reconsideration request.
Conflict of interest: JC fees from Pfizer, Novartis and Ipsen. JPC fees for
Novartis and Ipsen. AS fees from Pfizer, Novartis and Ipsen. RS fees from
Novartis and Ipsen. Again, this gives the same limited endorsement for NETest
that reference #21 in the policy gives.

Koenig et al. (171) The authors analyzed 62 patients with neuroendocrine
neoplasms of the colon and rectum. However, it appears that NETesting was not
done on any of these patients. This paper does not support NETest.

Marotta et al. (172) The authors reviewed the literature about chromogranin A
and studies involving NETest. We believe all these studies have been reviewed
for this LCD. The authors concluded, "Hence, the new frontier seems to be
represented by multianalyte approaches. Particularly, a blood-based algorithm
including simultaneous determination of 51 NEN-specific markers has been
developed in recent years(Modlin et al.2013) and all comparative studies were
concordant in reporting significantly better metrics as compared with CgA
(Modlin et al. 2014a,b, 2015, 2016, Bodei et al. 2016, Pavel et al.2017a)."
They also note that current indications from the ENETS and other major
societies dealing with NENs, which still recommend broad spectrum use of
circulating CgA for the diagnostic definition. In addition, this paper does
not show any improved survival for patients using NETest. The declaration of
interest was no conflicts. Since the authors note that specialty societies
have not accepted NETesting and that they do not note any increased survival
in using NETesting, and they do not show any independent validation, this
paper gives minimal support to NETest.

Yur R. (173) This is "in press." Draft, unpublished literature and not
appropriate to review. This is not acceptable medical literature and cannot be
reviewed. It does not support NETest.

Appetecchia et al. (174) The authors reviewed the literature, and conclude
that chromogranin A (CgA) is not a good biomarker for neuroendocrine tumors.
They then note, "An mRNA-based, specific multianalyte assay with algorithmic
analyses has been shown to have better sensitivity and specificity than CgA in
initial clinical studies." The first reference given for this statement is
"Modlin IM, Drozdov I, Kidd M. The identification of gut neuroendocrine tumor
disease by multiple synchronous transcript analysis in blood. PLoS One.
2013;8(5):e63364" which is reference # 63 in the LCD. This was also reviewed
by this contractor in a prior reconsideration and not felt to adequately
support NETest. The second of two references is "Modlin IM, Drozdov I, Alaimo
D, et al. A multianalyte PCR blood test outperforms single analyte ELISAs
(chromogranin A, pancreastatin, neurokinin A) for neuroendocrine tumor
detection. Endocr Relat Cancer. 2014;21(4):615-628" which is reference #29 in
the policy. In addition, this paper also has been previously reviewed by this
contractor in a prior reconsideration request. Upon review both times, neither
were felt to give adequate support of NETest. The issue for NETest is whether
peer-reviewed medical literature found on the US National Library of Medicine,
National Institutes of Health shows that NETest results in improved outcomes.
The authors note that the studies cited were initial clinical studies. Further
studies including independent clinical verification are need. This paper does
not give any new support to NETest.

Auernhammer et al. (175) This paper was submitted twice by Wren Laboratories.
Please see #112 above.

Zatelli M. (176) This paper was submitted twice by Wren Laboratories. Please
see # 157 above.

Muffatti et al. (177) This appears to be a teaching case report in a book
concerning a patient with a neuroendocrine tumor and discussion also on
localization. The authors do comment on NETest. "... and novel biomarkers with
improved predictive capability are currently in advanced clinical development.
Among them, there are circulating tumor cells (CTCs) and multianalyte whole
blood RNA signature (NETest). Changes in CC count have been recently
associated with treatment response and survival but treatment personalization
based on CT7Cs is currently not feasible for PanNENs. On the other hand, the
NETest has shown impressive results in terms of sensitivity and specificity
and its posttreatment changes seem to accurately predict response to operative
resection and PRRT. However, similarly to CTCs, treatment personalization
based on NETest baseline value has never been evaluated so far. Both CTCs and
NETest might be useful for follow-up in the adjuvant setting, although
clinical validation is still needed." There are not conflict of interest
disclaimers. This paper does mention some positive features of NETest but does
not support that it is established as a proven biomarker or that it improves
overall survival. Thus it does not currently support NETest.

Khatami et al. (178) This review paper does mention NETest in paragangliomas
and pheochromocytomas. It cites a number of papers that have previously been
reviewed for the LCD. These studies also have not shown any improvement in
overall survival. This paper does not give robust support for NETest.

La Rosa et al. (179) This review covers many aspects of these tumors. It does
note that "The multianalyte-derived NET gene signature encompasses the
expression of 51 genes which are assessed by four different prediction
algorithms and seem to give information on tumor state and evolution from
stability to progression. This approach defines the circulating fingerprint of
the tumor showing a higher sensitivity and specificity that traditional
secretory markers. The gene expression profile is mathematically analyzed
using specific algorithms, which define tumor activity. Interesting, recent
data have suggested that Circulating Transcript Analysis (NETest) may identify
tumor categories with a different prognosis and response to somatostatin
analogues and peptide receptor radionuclide (PRRT) therapy. However, this new
approach shows some problematic issues including technical complexity, which
restricts the analysis to specific laboratories." It also cites the Delphic
consensus which has been included in the LCD as reference #24 (Oberg K,
Krenning E, Sundin A, et al. A Delphic consensus assessment: imaging and
biomarkers in gastroenteropancreatic neuroendocrine tumor disease management.
Endocr Connect. 2016;5(5):174-187.) The acceptability of this is dampened by
the fact that this conference was supported by Clifton Life Sciences, which
owns Wren Laboratories, and eight of the sixteen panelists are listed as
having financial ties to Wren Laboratories. There are no disclaimers. This
publication does not offer robust support for NETest. It does not mention any
long term follow-up studies, improved overall survival or independent,
external validation of the results.

Holdenrieder et al. (180) This review covers many aspects of these tumors. The
author notes, "A consensus paper on the use of biomarkers for NET disease
outlined the need for circulating biomarkers for diagnosis, prognosis,
monitoring therapy response, identifying minimal residual disease, and
detection of recurrent disease. While the limitations of monoanalyte in
sensitivity and specific for GEP and lung NETs was recognized, more accurate
diagnostic tools were looked for. Current research approaches address
circulating DNA, mRNA, microRNA, and metabolomics biomarkers as well as
circulating tumor cells; however, their clinical utility still has to be
proven." The author declared no conflict of interest. We agree that clinical
utility must be proven. That includes prolongation of overall survival, and of
course independent, external validation of the results. In addition, the
problems with consensus has been noted multiple times in these reviews. Thus
at present, this is not robust support for NETest.

Rindi et al. (181) NGS really appreciates this editorial being sent. Since it
is an editorial (but with many NETest references) we did not find it on our
search for NETest support. These prominent experts give an unbiased evaluation
of the paper Liu, E. et al. Assessment of NETest Clinical Utility in a U. S.
registry-based Study. Oncologist
https://doi.org/10.1634/theoncologist.2017-0623 (2018). "In this study, 100
patients were monitored for 6-12 months. Patients received the test at
enrolment, were assigned to two groups depending on whether they had stable
disease ('watch-and-wait' group) or progressive disease ('intervention' group)
according to Response Evaluation Criteria In Solid Tumors (RECIST) criteria,
and separately analyzed. Seventy-two patients received a single test, and the
rest received 2-4 tests. The data provided suggest tumour progression can be
predicted earlier using NETest than with established RECIST criteria" However,
Rindi and Wiedenmann noted, "The presented data, however, must be considered
with caution, given the focus of the study on neuroendocrine cancer.
Neuroendocrine neoplasia encompasses distinct tumour entities with growth
behaviors that vary from extremely slow to very rapid. The incidence and
aggressiveness of neuroendocrine neoplasms is highly dependent on the
anatomical site of origin. The most aggressive disease is frequently found in
the pulmonary tract (in the form of small-cell lung cancer [SCLC], and is also
referred as neuroendocrine carcinoma (NEC) when found at other anatomical
sites. The most indolent cases are frequently detected in the digestive system
were previously termed carcinoids and are currently referred to as
neuroendocrine tumours (NETs).The introduction of standard grading and staging
tools for cancer in routine practice in the past decade greatly improved our
knowledge of neuroendocrine neoplasia. Necessarily, inclusion criteria
(according to anatomical site, and staging and grading parameters) need to be
accurately defined before conducting studies in patients with neuroendocrine
cancer in order to achieve a clear understanding of the results. Liu and co-
workers focused their attention on patients with NET (97 of 100 patients
tested). The most aggressive poorly differentiated forms of neuroendocrine
cancer were also represent, but only in a very small subgroup (1 patient had
SCLC and 2 patients had poorly differentiate NECs). The investigators used
NETest to evaluate blood samples of patients who predominantly had advanced-
stage neuroendocrine cancer (96 patients had stage IV disease), but with
variable disease grades, mostly, low grade (45 of 86 patients) or intermediate
grade (37 of 86 patients), and originating from different organs (68% in the
digestive tract and pancreas)....The emerging picture in this study of 100
patients is promising, but not all the results aligned with the expectation.
Discordance emerged when the NETest did not correlate with either absence or
presence of the disease (4%of patients) or with stable or progressive disease
status (17%and 23% of patients respectively) determined with stand methods. In
36% to 38% of patients in both groups the use of the test led to changes in
patient management, resulting in a reduction of the number of imaging
procedures performed. "Unfortunately, the specificity and sensitivity of the
test were not provided nor could they be accurately calculated with the data
made available; however, the overall concordance with disease status reported
(85) was well below 100%, a value necessary to ensure that not a single
patient is misdiagnosed and therefore denied the most appropriate treatment.
Understandably, reaching perfection is almost impossible in the real word, and
this principle holds true especially in the complex management of patients
with neuroendocrine neoplasia. Nevertheless, a clear definition of the
sensitivity and specificity of NET is needed before the assay is widely
adopted in clinical practice. Furthermore, whether the test performs
identically regardless of tumour grade, tumour volume and/or treatment history
remains unclear--the presented patient numbers are too small and the selected
cohort was to heterogeneous...Thus, the cost-benefit radio needs to be
accurately established once NETest has been studied in larger and homogenous
populations of patients with neuroendocrine cancer" [note: Medicare does not
use cost as a decision for coverage]. Competing interests: G.R. has received
honoraria from Ipsen and Novartis for is role in their speaker's bureau. B.W.
declares no competing interests." This paper refutes the contention that
NETest is proven. This paper does not support NETest.

Chen et al. (182) This paper was submitted twice by Wren Laboratories. Please
see #76 above.

Liu et al. (183) This paper was submitted twice by Wren Laboratories. Please
see #77 above.

Lybaert et al. (184) This paper does not appear to mention NETest. It does not
support NETest

Guzman et al. (185) This article is in Spanish. It is not acceptable support
for NETest.

Kyriakopoulos et al. (186) This paper was submitted twice by Wren
Laboratories. Please see #111 above.

  * Some of the publications reviewed for NETest are favorable. A few suggest it has potential but needs more independent studies and validation before it can be recommended. Most do not mention NETest at all as an option in diagnosing and/or treating neuroendocrine tumors. All the favorable publications have been authored or co-authored by Modlin & Kidd. Perhaps this is to be expected for a proprietary laboratory developed test, but it nonetheless means there has been no independent validation of the test. Such proprietary laboratory developed tests can be developed with Federal Drug Administration (FDA) review and clearance. Unfortunately, the FDA has no "final guidance on the oversight of laboratory developed tests" (73).
  * As noted above, all of the favorable papers have Dr. Irvin Modlin and/or Dr. Mark Kidd as authors. Equally important is the fact that Dr. Kidd is an employee of Wren Laboratories, which has the proprietary rights to NETest. Dr. Modlin is listed on most of the papers as a consultant for Wren Laboratories. On some, he is listed as an employee of Wren Laboratories. Many of the other papers have other co-authors who are listed as employees of Wren Laboratories. Many of the articles are sponsored by Clifton Life Sciences, which is the parent company of Wren Laboratories. There is much perceived conflict of interest that appears to permeate through these publications.
  * To make these papers on NETest more credible, an independent, external validation must be performed. In particular, some of the independent review articles also called for "confirmation" of promising results. This has not happened yet.
  * Related, independent societies and organizations such as National Comprehensive Cancer Network (NCCN) have not yet recommended the NETest. The only two such endorsements were by Oberg K, Modlin I, et al: _Consensus on Biomarkers for Neuroendocrine Tumour Disease_ and by Oberg, Eric Krenning: _Delphic consensus assessment: imaging and biomarkers in gastroenteropancreatic neuroendocrine tumor disease management_. Dr. Modlin was the second author of this first consensus. "The experts were of the opinion that such laboratory tests should be initially undertaken at a single central laboratory."  This was supported by Clifton Life Sciences, which owns Wren Laboratories, and eight of the sixteen panelists are listed as having financial ties to Wren Laboratories. The second consensus also had multiple panelists associated with Wren Laboratories. Thus, there is potential concern about how impartial these two recommendations are.
  * The favorable papers, all from Wren Laboratories, claim that NETest can detect disease recurrence as long as one year before radiographic evidence of disease. Perhaps this is true. However, are there any clinical ramifications as a result of this finding? Will there be some type of treatment based on detection of this marker of disease which will result in improved outcomes? Unfortunately, there is currently no such evidence.
  * Related, this test only offers prognostic information, and its predictive value is neither solid nor associated with improved survival.
  * The evidence for clinical utility is limited, the documentation about analytical and clinical validity is also limited.
  * Most important, as noted above, none of the articles show any improved overall survival. If beneficiaries do not live longer, the value of the test is questionable.
  * One can argue that this kind of "black box" assay that incorporate multiple genetic markers, particularly RNA, into a proprietary calculation that generates some kind of dimensionless risk index, should have a higher burden of proof than individual gene or gene panel tests. With individual gene testing for many cancers, the results of the testing results in a targeted medical treatment (e.g. BRAF testing). With an elevated NETest, one does not know if a specific therapeutic intervention is indicated.