Table 1. PathFinderTG Tests (4) Test Description Specimen Type(s) PathFinderTG Pancreas

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1 Subject: PathFinderTG Molecular Testing Page: 1 of 15 Last Review Status/Date: September 2015 PathFinderTG Molecular Testing Description The patented PathFinderTG test is a molecular test to be used adjunctively in cases when a definitive pathologic diagnosis cannot be made, because of inadequate specimen or equivocal histologic or cytologic findings. RedPath Integrated Pathology (Pittsburg, PA), the test provider, states that PathFinderTG produces mutational profiles to help physicians resolve complex diagnostic dilemmas in patients who are at risk of cancer. Background Topographic genotyping (TG), also called molecular anatomic pathology, integrates microscopic analysis (anatomic pathology) with molecular tissue analysis. Under microscopic examination of tissue and other specimens, areas of interest may be identified and microdissected to increase tumor cell yield for subsequent molecular analysis. TG may permit pathologic diagnosis when first-line analyses are inconclusive. (1) RedPath Integrated Pathology has patented a proprietary platform, called PathFinderTG, to provide mutational analyses of patient specimens. The patented technology permits analysis of tissue specimens of any size, including minute needle biopsy specimens, and any age, including those stored in paraffin for over 30 years. (2) RedPath currently offers 5 PathFinderTG tests (listed and briefly described in Table 1). As stated on the company web site, PathFinderTG integrates molecular analyses with first-line results (when these are inconclusive) and pathologist interpretation to provide clinically valid and useful diagnostic and prognostic information. (3) Although the web site states that PathFinderTG is clinically validated as reported in over 200 peer-reviewed articles, test performance information is not provided. Table 1. PathFinderTG Tests (4) Test Description Specimen Type(s) PathFinderTG Pancreas Uses loss of heterozygosity markers, oncogene mutations, and DNA content abnormalities to stratify patients according to their risk of progression to cancer Pancreatobiliary fluid/ercp brush, pancreatic masses, or pancreatic tissue PathFinderTG Measures the presence and extent of genomic instability Esophageal tissue Barrett PathFinderTG Biliary and integrates those results with histology Uses oncogene mutations and loss of heterozygosity markers to identify whether patients with biliary strictures have a malignant neoplasm or benign reactive disease Biliary brush/supernatants

2 Subject: PathFinderTG Molecular Testing Page: 2 of 15 PathFinderTG Metastases versus Primary Tumors PathFinderTG Glioma Uses molecular markers and mutations to determine whether concurrent tumors are synchronous primaries or metastatic disease Intended to help differentiate between gliosis and glioma, grade of glioma, and type of malignancy ERCP, endoscopic retrograde cholangiopancreatography Slides Not reported Management of Mucinous Neoplasms of the Pancreas In 2012, an international consensus panel published consensus statements for the management of IPMN and mucinous cystic neoplasm (MCN) of the pancreas. (5) These statements were based on a consensus symposium held in Japan in 2010 and updated a 2006 publication by this same group. (6) The panel recommended surgical resection for all surgically fit patients with main duct IPMN or MCN. For branch duct IPMN, surgically fit patients with cytology that is suspicious or positive for malignancy are recommended for surgical resection, but patients without high-risk stigmata or worrisome features may be observed with surveillance. High-risk stigmata are: obstructive jaundice in proximal lesions (head of the pancreas); presence of an enhancing solid component within the cyst; or 10 mm or greater dilation of the main pancreatic duct. Worrisome features are: pancreatitis; lymphadenopathy; cyst size 3 cm or greater; thickened or enhancing cyst walls on imaging; 5 to 10 mm dilation of the main pancreatic duct; or abrupt change in pancreatic duct caliber with distal atrophy of the pancreas. Regulatory Status These patented diagnostic tests are only available through RedPath Integrated Pathology (Pittsburgh, PA). The PathFinderTG Molecular Test is not subject to review by the U.S. Food and Drug Administration (FDA) because it is a laboratory-developed test (LDT) conducted only at RedPath Integrated Pathology s licensed laboratory. Laboratories performing LDTs must be licensed for highcomplexity testing under the Clinical Laboratory Improvement Amendments of 1988 (CLIA). RedPath is licensed under CLIA. Related Policies None Policy *This policy statement applies to clinical review performed for pre-service (Prior Approval, Precertification, Advanced Benefit Determination, etc.) and/or post-service claims. Molecular testing using the PathFinderTG system is considered investigational for all indications including the evaluation of pancreatic cyst fluid and suspected or known gliomas and Barrett esophagus. Rationale RedPath Integrated Pathology offers the PathFinderTG mutational profiles in an attempt to help physicians resolve complex diagnostic dilemmas in patients who are at risk of cancer (see Appendix

3 Subject: PathFinderTG Molecular Testing Page: 3 of 15 Table 1 for genetic testing categories). Because of the scope of claims by the company for widespread applications of PathFinder TG in multiple organ systems and clinical scenarios, and more than 200 peer-reviewed articles that have clinically validated the test, (3) we chose 3 representative applications to address in this policy-pancreatic cysts, gliomas, and Barrett esophagus. Published studies reviewed for this policy include those cited by RedPath website as well as those that represent current understanding of molecular profiles of various tumor types and their potential role in clinical decision making. Pancreatic Cysts The diagnosis of cystic pancreatic lesions is usually performed by endoscopic, ultrasound-guided fineneedle aspiration sampling of the fluid and cyst wall for cytologic examination and analysis. Cytologic examination of these lesions can be difficult or indeterminate due to low cellularity, cellular degeneration, procedural difficulties, etc. Ancillary tests (eg, amylase, lipase, carcinoembryonic antigen levels) often are performed on cyst fluid to aid in the diagnosis, but results still may be equivocal. Information provided by additional testing modalities would, therefore, be potentially useful. As shown in Table 1, the PathFinderTG Pancreas test uses loss of heterozygosity (LOH) markers, oncogene mutations, and DNA content abnormalities to stratify patients according to their risk of progression to cancer. (4) Evidence From a convenience sample of tissue specimens archived between 1999 and 2004, Lapkus et al (2006) described mutations in 40 pancreatic duct and 21 bile duct brushing specimens. (7) No description of patients or tumor stage was reported. Cells were diagnosed by conventional cytology as malignant (n=10), atypical (n=20), or benign (n=10) and subsequently examined for mutations in genomic markers commonly associated with tumor suppressor gene loss and oncogene activation in pancreatic and biliary cancer (eg, LOH for a panel of 15 markers [1p, 3p, 5q, 9p, etc.]) and point mutation in KRAS-2. The authors suggested [k]nowing that cancer evolves through a temporal process of mutation acquisition raises the question whether this sequence can be determined in clinical specimens. Mutations appearing with greater frequency were therefore considered to have been acquired earlier. The authors reported the most commonly-occurring mutations (eg, KRAS2 point mutation and LOH for 3p25-26, 17q21, and others in pancreatic cytology; LOH for 17p13, 1p36, and other for biliary cytology). Tissue histologic findings were available for 4 samples in the malignant group (all carcinomas), 4 in the benign group (3 were benign), and 9 in the atypical group (4 carcinomas). These results provided little evidence to support clinical value of the mutation patterns. Results from just 17 of 40 samples were compared to tissue diagnoses; therefore, measures of accuracy are not calculable. The cross-sectional study design further prevented meaningful conclusion. Although these findings suggest a possible sequence of mutation acquisition in pancreatic and biliary ductal carcinoma, they are not definitive due to the small sample size and lack of replication. Further, how identification of early oncogenic mutations may be incorporated into diagnosis and treatment planning is yet undetermined. Khalid et al (2004) reported results from a retrospectively assembled case series using LOH in the diagnosis of pancreatic malignancy. (8) In an apparent convenience sample of 26 patients with either

4 Subject: PathFinderTG Molecular Testing Page: 4 of 15 surgical removal (n=25) or long-term follow-up (n=1), there were 17 cancers (6 pancreatic and 11 cholangiocarcinomas) and 9 non-cancers. No description of patients or tumor stage was reported. Using standard microscopic analysis of the cytology specimens, 8 were judged malignant, 10 were indeterminate, and 8 were benign. Molecular tests (eg, LOH, KRAS) were conducted on cytology and tissue specimens. PathFinderTG sensitivity for a diagnosis of malignancy was 100% (95% confidence interval [CI], 83 to 100), and specificity was100% (95% CI, 70 to 100; CIs calculated). Whether molecular findings would have had clinical impact was unclear. In addition, results were not presented in a manner that allowed direct comparison between molecular and standard testing, both for the cytology and the tissue specimens. In a case series, Khalid et al (2005) prospectively collected fluid from 116 pancreatic cysts. (9) Molecular analyses were restricted to 31 patients who underwent surgery and 5 patients [who] reached a final diagnosis of malignancy based on cytology. In this subgroup of 36, molecular analyses included KRAS point mutation and a broad panel of LOH markers. Whether those conducting molecular assays were blinded to confirmatory results was not reported. Cutoff points defining significant LOH at loci were established using normal specimens; standard deviation greater than 2 was defined as abnormal. Sensitivity and specificity of KRAS mutation alone for distinguishing premalignant malignant cysts were 93% (95% CI, 62 to 100) and 87% (95% CI, 62 to 96; CIs calculated). Subsequent testing for LOH did not alter sensitivity and correctly reclassified 1 false positive result, increasing specificity to 93% (95% CI, 70 to 100). Analysis from this small sample did not demonstrate incremental value for LOH testing added to KRAS. These studies highlight important issues pertaining to test accuracy and whether there is evidence that PathFinderTG results provide incremental information that informs diagnostic and prognostic decision making for patients with pancreatic cysts, including the following: 1. whether progression of genetic mutations is sufficiently defined to use for diagnosis and prognosis 2. sampling variability of fluid and tissue specimens 3. study design and conduct including patient (sample) selection and blinding of personnel performing assays to clinical outcomes 4. small sample sizes and absence of sufficient tissue samples to compare with PathFinderTG results for a substantial proportion of patients 5. lack of adequate patient follow-up and validation/replication of findings First, stepwise accumulation of KRAS mutations and allelic losses (LOH) accompanying cancer progression in pancreatic ductal adenocarcinomas have been found. (10, 11) However, overlap between molecular findings in inflammatory and neoplastic conditions has also been described, specifically KRAS mutations in chronic pancreatitis. (12-15) Progression of genetic mutations in mucinous pancreatic tumors has been described and, although there appear to be similarities with pancreatic ductal adenocarcinomas, the precise pattern and progression of these mutations remain to be elucidated. For example, while suggesting the pattern of KRAS mutation followed by allelic loss predicts malignancy in pancreatic cysts, Khalid et al (2005) also noted a lack of clarity in the pattern and rate of mutation accumulation. (9)

5 Subject: PathFinderTG Molecular Testing Page: 5 of 15 Second, potential sampling discrepancies between the molecular profile of a pancreatic cyst wall aspirate or fluid and tissue specimen needs to be further addressed. This is particularly important in mucinous cystic neoplasms (MCNs) of the pancreas, which may show differing histologic features in different areas of the tumor and may have focal invasion. (16) To further complicate this issue, RedPath does not address the genetic heterogeneity seen among the subtypes of MCNs (for example, KRAS mutations have been described as occurring in 20% of mucinous cystic adenomas, 33% of adenomas with moderate dysplasia, and 89% with carcinoma in situ). (16) Third, whether those performing the assay were blinded to surgical results was not stated in the studies reviewed. Clear description of case selection (eg, consecutive or convenience) was absent. Fourth, sample sizes were small, as reflected in the wide confidence intervals calculated for reported sensitivities and specificities. Finally, both adequate follow-up and replicated results (validation) are required to demonstrate whether PathFinderTG results inform diagnostic and prognostic decision making in a manner that will benefit patients. We are unaware of long-term studies with defined clinical outcomes using PathFinderTG to differentiate neoplastic pancreatic cysts from those determined by the test as benign, implying that progression to cancer is improbable. However, international consensus recommends surgical resection for all surgically fit patients with MCN or main duct intraductal papillary mucinous neoplasm (IPMN). (5) This is due to the uncertainty of the natural history of MCN and main duct IPMN and the presumed malignant potential of all types. (17-19) Khalid et al (2009) published results of a prospective multicenter study to validate their initial results from a single center pilot study, (9) and to evaluate the role of pancreatic cyst fluid DNA analysis in differentiating mucinous from nonmucinous cysts. (20) Patients were seen for endoscopic ultrasound (EUS)-guided evaluation of a pancreatic cyst between July 2004 and June 2006 at 1 of 7 participating centers. DNA analysis was performed only in cases that reached conclusion (defined as surgical resection of the lesion or a diagnosis of malignancy based on fine-needle aspiration cytology). DNA analysis comprised direct sequencing of the first exon 1 of the KRAS gene and polymerase chain reaction (PCR) amplification of individual microsatellite markers (ie, variable number tandem repeats) to determine allelic imbalance (reported as allelic loss amplitude [ALA]). A total of 391 patients were enrolled; after exclusions for various reasons including no cyst seen by EUS or insufficient cyst fluid, 299 patients remained of which 124 of whom reached a final pathologic diagnosis based on surgical resection (n=98) or malignant cytology (n=26). An additional 11 cases were excluded for various pathologic diagnoses not included in the study methods (eg, islet cell tumors). The remaining 113 cysts were classified as benign nonmucinous (n=25) or mucinous (n=88). Of the mucinous, 40 were malignant and 48 were premalignant. Cyst fluid KRAS mutation with allelic loss showed a specificity of 96% for the diagnosis of malignancy but low sensitivity (37%). Optimal cutoff points for ALA were established and, at the level set, the sensitivity and specificity for distinguishing nonmucinous from mucinous cysts were 67% and 66%, respectively. A separate ALA cutoff value for malignancy yielded a 90% sensitivity and 67% specificity. Limitations of the study were acknowledged. These included lack of investigator blinding to the results of the DNA analysis and potential selection bias, as only lesions with confirmed pathologic findings were included. Therefore, the study sample comprised of a greater proportion of patients with malignant cysts than would have been expected.

6 Subject: PathFinderTG Molecular Testing Page: 6 of 15 In 2010, a systematic review of LOH-based topographic genotyping with PathFinderTG was prepared for the Agency for Healthcare Research and Quality (AHRQ) technology assessment program. (1) Key questions addressed the published evidence on the analytic test performance, diagnostic ability, and clinical validity of the test and what evidence there is comparing the PathFinderTG test with conventional pathology. Conclusions were that no studies included in the systematic review directly measured whether using LOH-based topographic genotyping with PathFinderTG improved patientrelevant clinical outcomes and that the eligible studies on the diagnostic and prognostic ability of the test were small in sample size, had overt methodologic limitations, and all but 1 performed retrospective assessments. The review pointed out that studies did not provide important information on patient selection, patient characteristics, treatments received, clinical end point definitions, justification of sample size, selection of test cut points, and selection among various statistical models. In addition, reviewers noted that there were strong indications that the selection of certain test cut points was determined post-hoc, in that cutoffs varied widely across studies and were not validated in an external population. Siddaqui et al (2013) examined cyst fluid DNA in 25 patients with pancreatic cysts diagnosed on imaging who underwent EUS-guided fine needle aspiration followed by surgical resection. (21) Cyst fluid DNA was analyzed for mutations in KRAS and guanine nucleotide binding protein, alphastimulating complex locus (GNAS). Nine cysts were classified by surgical pathology as intraductal papillary mucinous neoplasms. Sensitivity and specificity were, respectively, 67% and 69% for KRAS mutation, 44% and 100% for GNAS mutation, and 100% and 69% for both together. In comparison, sensitivity and specificity were, respectively, 44% and 100% for cytology, and 56% and 69% for CEA greater than 192 ng/ml. Larger prospective trials are needed to validate these findings. In 2011, RedPath Integrated Pathology established the National Pancreatic Cyst Registry, (22) and in 2015, published results of 492 (26%) registered patients. (23) The Registry website describes the registry as a prospective study to evaluate the performance characteristics and clinical utility of integrated molecular pathology and determine the predictive value of both traditional first-line tests and integrated molecular pathology. Ten academic medical centers and community-based practices registered patients who had pancreatic cysts, underwent PathFinderTG testing, and were followed for development of malignancy. Benign outcomes included benign surgical pathology results, low- or intermediate-grade dysplasia, resolution of cyst, or clinical follow-up by imaging for a minimum of 23 months without evidence of malignant outcome; malignant outcomes were determined by surgical pathology diagnosis of high-grade dysplasia, carcinoma in situ, or adenocarcinoma, newly diagnosed malignant cytology results, clinically confirmed pancreatic cancer in patient records, or death attributed to pancreatic cancer. Investigators compared the diagnostic performance of PathFinderTG to that of an international consensus classification scheme.5 Both classification schemes categorize patients with pancreatic cysts as high or low risk for malignancy; those considered high risk undergo surgical resection, and those considered low risk may elect observation with surveillance. At median follow-up of 35 months (range, 23-92), 66 patients (35%) were diagnosed with malignancy. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 83%, 91%, 58%, and 97% for PathFinderTG versus 91% (p=0.17), 46% (p<0.001), 21% (p<0.001), and 97% (p=0.88) for international consensus classification. A strength of the study is the inclusion of both surgery and surveillance groups. Limitations include the retrospective design, resulting in the exclusion of 74% of all registered patients due primarily to insufficient follow-up; relatively short follow-up for observing

7 Subject: PathFinderTG Molecular Testing Page: 7 of 15 malignant transformation of benign lesions; and the exclusion of patients classified as malignant by international consensus criteria who would not have undergone PathFinderTG testing. Because of these limitations, it is unclear whether classification by PathFinderTG improves treatment decision making and health outcomes, such as overall survival, compared with current classification criteria. Deftereos et al (2014) analyzed DNA in cell-free cytocentrifugation supernatant fluid from fine needle aspirates of solid pancreatic masses. [(24, 25) Investigators compared cell-free DNA with cellular DNA from microdissected stained cytology slides. Twenty-three specimens were classified cytologically as pancreatic carcinoma (n=5), mucinous neoplasm (n=5), pancreatitis (n=5), neuroendocrine neoplasm (n=1), negative (n=4), or suboptimal (paucicellular or acellular; n=3) and confirmed by histological examination of surgical or biopsy specimen. Mutational analyses of microdissected cell specimens and supernatant obtained from every specimen were compared. Mutational analyses comprised KRAS codon 12 point mutations and LOH of 16 microsatellite markers. Three cell samples failed PCR amplification for 1 or both analyses (KRAS or LOH); all supernatant DNA was amplifiable. Excluding suboptimal cytological specimens, sensitivity and specificity of KRAS plus LOH testing for detecting malignancy were both 100% for supernatant DNA, and 89% and 100%, respectively, for cellular DNA. The study suggests a possible additional source of DNA for molecular analysis, but results require replication in larger studies showing impacts on clinical decision-making. Two studies attempted to show the incremental value of molecular analysis of pancreatic cyst DNA (from cytology slides or aspirated fluid) to cytologic diagnosis of malignancy. Gillis et al (2015) in Ireland conducted a systematic review of the literature but found sufficient evidence to evaluate incremental value of KRAS mutational status only. (26) Evidence for LOH and DNA quantification was insufficient to form conclusions. Malhotra et al (2014) at RedPath retrospectively evaluated 30 patients who presented with pancreaticobiliary masses and had minimum follow-up of 3 months.27 Only 26 patients with a cytologic diagnosis of atypical, negative, or indeterminate underwent PathFinderTG mutational profiling, precluding assessment of diagnostic performance. Cytology correctly diagnosed 4 of 21 malignant cases (sensitivity, 19%), and identified 7 of 9 patients with nonaggressive disease (specificity, 78%). PathFinderTG correctly diagnosed 8 of 17 malignant cases (sensitivity, 47%), and identified all 9 patients with nonaggressive disease (specificity, 100%). Although the combination of positive cytology and positive PathFinderTG results improved sensitivity to 57% (12/21), 9 malignant cases were missed by both tests. Two other studies from RedPath yielded inconclusive results due to their retrospective design and short follow-up (median, 27 months), use of a 3-tier classification scheme rather than the 4-tier classification currently used for PathFinderTG, and diagnostic performance of uncertain clinical usefulness (sensitivity, specificity, PPV, and NPV of 75%, 88%, 82%, and 81%, respectively). (28, 29) Section Summary: The evidence reviewed does not demonstrate that PathFinderTG has incremental clinical value for diagnosis or prognosis of pancreatic cysts and associated cancer. Gliomas Limitations of pathologic examination for diagnosing and grading gliomas have been described. (30, 31) This is partly due to the small brain biopsy sizes, as well as to interobserver variability between pathologists. In addition, different glial tumors may respond differently to therapy. Defining prognosis

8 Subject: PathFinderTG Molecular Testing Page: 8 of 15 and predicting therapeutic response is therefore of considerable interest. The PathFinderTG test is proposed to address these issues using panels of LOH markers from stereotactic brain biopsies or fineneedle aspirations to: provide glioma diagnosis and classification assess tumor aggressiveness (discriminating between low- and high-grade gliomas) discriminate glioma from reactive gliosis predict tumor response to certain chemotherapeutic regimens through analysis of the chromosomal 1p/19q deletion and identified LOH mutations Evidence In a 2004 study cited as clinical validation for PathFinderTG, investigators retrospectively analyzed archived tissue from 197 gliomas, performing polymerase chain reaction (PCR) for a panel of 12 to 16 LOH markers. (32) Loss of 1p and 19q were analyzed in all cases, with a subset (n=93) undergoing an extended panel of 16 markers. The authors reported, The qualitative and quantitative aspects of glioma allelic loss were correlated with the clinical impression of treatment response [to procarbazine, CCNU, vinblastine] as determined by imaging studies. Treatment response was classified as none, partial (25% decrease in size with stable or decreasing steroid dose), or complete ( disappearance of all contrast-enhancing tissue for gliomas and resolution of abnormalities on FLAIR sequences for lowgrade gliomas ). However, tables reported major response (undefined) rather than complete response. Of 96 patients with high-grade gliomas, 49 had follow-up of 6 months or longer and were assessed for therapy response: 11 had no response, 17 partial, and 21 major response; deaths occurring prior to 6 months were not reported. Predictive values (calculated from reported data) of at least partial response for 1p loss were positive predictive value (PPV) 100% (95% CI, 89 to 100%) and negative predictive value (NPV) 65% (95% CI, 41 to 83%); for 19q loss, PPV was 82% (95% CI, 64 to 92%) and NPV was 21% (95% CI, 8 to 43%). It is unclear whether the molecular data results were blinded for the classification of treatment response. The retrospective design, lack of follow-up information for 47 patients (24%), and no report of any censoring due to mortality are significant study limitations. In a retrospective case series, Finkelstein et al (2004) described LOH to distinguish neoplasms from reactive gliosis. (33) The study included archival tissue from 15 patients with reactive gliosis and 54 patients with gliomas of varying histologic type and grade. Molecular analyses were conducted blinded to clinical diagnosis, and the results showed no LOH in any of the cases of gliosis. From the group of 54 patients, 14 (74%) of 19 well-differentiated neoplasms showed allelic loss, 35(100%) of 35 highgrade gliomas showed at least 1 allelic loss alteration (33 [94%] of 35 had > 1 loss). Within this same study, investigators then analyzed LOH in an additional 16 cases with indeterminate histopathology (reactive gliosis vs. gliomas). Of 16 indeterminate cases, 9 (54%) were subsequently determined to be neoplastic based on clinical follow-up; LOH correctly identified 8 of these 9; sensitivity 89% (95% CI, 56 to 99%) and specificity 88% (95% CI, 53 to 99%). LOH correctly reclassified 3 (33%) of 9 as neoplasms initially diagnosed with benign disease, and 1 (14%) of 7 with benign disease initially diagnosed as neoplastic. While results were consistent with the study hypothesis, it will be necessary to apply the criteria determined in this study to additional validation samples. In addition, within this study, the sample size was small and confidence intervals were wide.

9 Subject: PathFinderTG Molecular Testing Page: 9 of 15 The only molecular test currently being performed on a relatively frequent basis for gliomas is the 1p/19q chromosomal loss for oligodendrogliomas. Yet optimal use of this test in the clinical care of patients remains unclear. (34) Although LOH involving multiple chromosomes has been observed in gliomas, and in some studies, LOH is predictive of tumor response to particular chemotherapeutic regimens; it is unclear that the information is meaningfully incremental to that provided by the 1p/19q data alone. (35) Also, some studies have shown conflicting results in terms of chemoresponsiveness for tumors with LOH involving chromosomes other than 1p/19q. (35) As noted by Lassman and Holland (2007): Current wisdom suggests that one of the main reasons for the poor responsiveness (of gliomas to therapy) is the existence of multiple genetic subsets of gliomas and the related lack of molecular stratification prior to treatment. Therefore, several strategies have been employed to divide gliomas into molecular subgroups that are either more or less responsive to existing therapies. Whether such stratification will result in effective therapies specific for each glioma subset is not yet clear. To what extent molecular subdivision of gliomas will be clinically useful remains to be seen. (34) Section Summary: The evidence reviewed does not demonstrate that PathFinderTG testing for diagnosis or predicting response to therapy provides a benefit to patients with gliomas. Barrett Esophagus The American Gastroenterological Association defines Barrett esophagus as replacement of normal epithelium at the distal esophagus by intestinal metaplasia, which predisposes to malignancy. (36) Although grading of dysplasia in mucosal biopsies is the current standard for assessing risk of malignant transformation, esophageal inflammation may mimic or mask dysplasia and interobserver variability may yield inconsistent risk classifications. (37) Additional prognostic information therefore may be potentially useful. Evidence Khara et al (2014) examined LOH in microsatellite regions of TP53 and CDKN2A tumor suppressor genes and in 8 other tumor suppressor genes (total 10 loci) as prognostic markers in Barrett esophagus. (38) Formalin-fixed paraffin-embedded (FFPE) tissues from 415 patients from 3 study sites who had histologically-diagnosed Barrett esophagus were microdissected to yield 877 specimens. Each was histologically classified as: normal squamous epithelium, columnar mucosa, intestinal metaplasia, indefinite for dysplasia (applied when cellular atypia is present but criteria for dysplasia are not met), low-grade dysplasia, high-grade dysplasia, or esophageal adenocarcinoma. At 1 study site, consensus diagnosis required agreement between 2 of 3 pathologists. All pathologists were blinded to molecular results, but it is unclear whether those conducting molecular analyses were blinded to pathology results. In molecular analysis, thresholds for defining significant LOH were determined using normal specimens; standard deviation greater than 2 was defined as LOH present. High clonality was defined as LOH mutation in more than 75% of DNA. Mutational load for each genomic locus was calulated by summing the proportional value of LOH and microsatellite instability (eg, 0.5 for low clonality, 1 for high clonality, 0.75 for microsatellite instability at a single locus, 0.5 for microsatellite stability at each additional locus). Mean mutational load increased with increasingly severe histology. Categories of

10 Subject: PathFinderTG Molecular Testing Page: 10 of 15 mutational load (none, low [lower 95 th percentile], and high [upper 5 th percentile]) appeared to discriminate less severe and more severe histology, but there was considerable overlap between now and low mutational load and between low and high mutational load. How these findings may be applied in clinical practice is unclear. Section Summary: The evidence reviewed does not demonstrate that PathFinderTG testing for prognosis of Barrett esophagus adds incremental value to current prognostic assessments. Ongoing Clinical Trials Some currently unpublished trials that might impact this policy are listed in Table 2. Table 2. Summary of Key Trials NCT No. Trial Name Planned Enrollment Ongoing NCT NCT: national clinical trial. The Diagnostic Yield of Malignancy Comparing Cytology, FISH and Molecular Analysis of Cell Free Cytology Brush Supernatant in Patients With Biliary Strictures Undergoing Endoscopic Retrograde Cholangiography (ERC): A Prospective Study Practice Guidelines and Position Statements Completion Date 110 Dec 2016 American Gastroenterological Association (AGS) In 2011, AGS published a medical position statement on the management of Barrett esophagus. (36) Based on findings from a technical review, (39) AGS suggest[s] against the use of molecular biomarkers to confirm the histological diagnosis of dysplasia or as a method of risk stratification for patients with Barrett s esophagus at this time (weak recommendation, low-quality evidence). National Comprehensive Cancer Network (NCCN) Current NCCN guidelines for pancreatic adenocarcinoma, (40) central nervous system cancers, (41) esophageal and esophagogastric junction cancers, (42), and hepatobiliary cancers (43) do not include recommendations for molecular anatomic pathology or integrated molecular pathology. U.S. Preventive Services Task Force Recommendations Not applicable Summary Evidence reviewed for 3 representative uses for this test has significant limitations, as discussed. Demonstrating the utility of a test for diagnostic and prognostic purposes or to predict therapeutic response requires that results accurately inform clinical decision making in a manner leading to a net health benefit defined by clinical outcomes. Results also must be clearly reproducible, as shown by applying the test (with priori-defined cutoff points) to independent samples for validation. Because the impact of this technology on net health outcomes is unknown and because outcomes with this

11 Subject: PathFinderTG Molecular Testing Page: 11 of 15 technology compared with existing alternatives (ie, incremental value) are unknown, the PathFinderTG testing is considered investigational for all indications, including evaluation of pancreatic cyst fluid, suspected/known gliomas, and Barrett esophagus. References 1. Trikalinos T, Terasawa T, Raman G. A systematic review of loss-of-heterozygosity based topographic genotyping with PathfinderTG. AHRQ Technology Assessment Program Available online at: Last accessed June U.S. Patent #7,014,999. Finkelstein et al. March 21, Topographic genotyping. Available online at: Last accessed June RedPath Integrated Pathology. Overview Available online at: Last accessed June RedPath Integrated Pathology. Services Available online at: June Tanaka M, Fernandez-del Castillo C, Adsay V, et al. International consensus guidelines 2012 for the management of IPMN and MCN of the pancreas. Pancreatology. May-Jun 2012; 12(3): PMID Tanaka M, Chari S, Adsay V, et al. International consensus guidelines for management of intraductal papillary mucinous neoplasms and mucinous cystic neoplasms of the pancreas. Pancreatology. 2006; 6(1-2): PMID Lapkus O, Gologan O, Liu Y, et al. Determination of sequential mutation accumulation in pancreas and bile duct brushing cytology. Mod Pathol. 2006; 19(7): Khalid A, Pal R, Sasatomi E, et al. Use of microsatellite marker loss of heterozygosity in accurate diagnosis of pancreatoticobiliary malignancy from brush cytology samples. Gut. 2004; 53(12): Khalid A, McGrath K, Zahid M, et al. The role of pancreatic cyst fluid molecular analysis in predicting cyst pathology. Clin Gastroenterol Hepatol. 2005; 3(10): Furukawa T, Sunamura M, Horii A. Molecular mechanisms of pancreatic carcinogenesis. Cancer Sci. 2006; 97(1): Koorstra JB, Hustinx SR, Offerhaus GJ, et al. Pancreatic carcinogenesis. Pancreatology. 2008; 8(2): Khalid A, Nodit L, Zahid M, et al. Endoscopic ultrasound fine needle aspirate DNA analysis to differentiate malignant and benign pancreatic masses. Am J Gastroenterol. 2006; 101(11): Muller P, Ostwald C, Puschel K, et al. Low frequency of p53 and ras mutations in bile of patients with hepato-biliary disease: a prospective study in more than 100 patients. Eur J Clin Invest. 2001; 31(3): Popovic HM, Korolija M, Jakic RJ, et al. K-ras and Dpc4 mutations in chronic pancreatitis: case series. Croat Med J. 2007; 48(2):

12 Subject: PathFinderTG Molecular Testing Page: 12 of Uehara H, Nakaizumi A, Tatsuta M, et al. Diagnosis of pancreatic cancer by detecting telomerase activity in pancreatic juice: comparison with k-ras mutations. Am J Gastroenterol. 1999; 94(9): Singh M, Maitra A. Precursor lesions of pancreatic cancer: molecular pathology and clinical implications. Pancreatology. 2007; 7(1): Khalid A, Brugge W. ACG practice guidelines for the diagnosis and management of neoplastic pancreatic cysts. Am J Gastroenterol. 2007; 102(10): Oh HC, Kim MH, Hwang CY, et al. Cystic lesions of the pancreas: challenging issues in clinical practice. Am J Gastroenterol. 2008; 103(1): Tanaka M, Chari S, Adsay V, et al. International consensus guidelines for management of intraductal papillary mucinous neoplasms and mucinous cystic neoplasms of the pancreas. Pancreatology. 2006; 6(2-Jan): Khalid A, Zahid M, Finkelstein SD, et al. Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study. Gastrointest Endosc. 2009; 69: PMID Siddiqui AA, Kowalski TE, Kedika R, et al. EUS-guided pancreatic fluid aspiration for DNA analysis of KRAS and GNAS mutations for the evaluation of pancreatic cystic neoplasia: a pilot study. Gastrointest Endosc. 2013; 77(4): PMID Redpath Integrated Pathology. National Pancreatic Cyst Registry. Available online at: Last accessed June Al-Haddad MA, Kowalski T, Siddiqui A, et al. Integrated molecular pathology accurately determines the malignant potential of pancreatic cysts. Endoscopy. Feb 2015; 47(2): PMID Deftereos G, Finkelstein SD, Jackson SA, et al. The value of mutational profiling of the cytocentrifugation supernatant fluid from fine-needle aspiration of pancreatic solid mass lesions. Mod Pathol. 2014; 27(4): PMID Finkelstein SD, Bibbo M, Kowalski TE, et al. Mutational analysis of cytocentrifugation supernatant fluid from pancreatic solid mass lesions. Diagn Cytopathol. Nov Gillis A, Cipollone I, Cousins G, et al. Does EUS-FNA molecular analysis carry additional value when compared to cytology in the diagnosis of pancreatic cystic neoplasm? A systematic review. HPB (Oxford). May 2015; 17(5): PMID Malhotra N, Jackson SA, Freed LL, et al. The added value of using mutational profiling in addition to cytology in diagnosing aggressive pancreaticobiliary disease: review of clinical cases at a single center. BMC Gastroenterol. 2014; 14:135. PMID Kung JS, Lopez OA, McCoy EE, et al. Fluid genetic analyses predict the biological behavior of pancreatic cysts: three-year experience. JOP. Sep 2014; 15(5): PMID Winner M, Sethi A, Poneros JM, et al. The role of molecular analysis in the diagnosis and surveillance of pancreatic cystic neoplasms. JOP. 2015; 16(2): PMID Aldape K, Burger PC, Perry A. Clinicopathologic aspects of 1p/19q loss and the diagnosis of oligodendroglioma. Arch Pathol Lab Med. Feb 2007; 131(2): PMID

13 Subject: PathFinderTG Molecular Testing Page: 13 of Coons SW, Johnson PC, Scheithauer BW, et al. Improving diagnostic accuracy and interobserver concordance in the classification and grading of primary gliomas. Cancer. 1997; 79(7): Mohan D, Finkelstein SD, Swalsky PA, et al. Microdissection genotyping of gliomas: therapeutic and prognostic considerations. Mod Pathol. 2004; 17(11): Finkelstein SD, Mohan D, Hamilton RL, et al. Microdissection-based genotyping assists discrimination of reactive gliosis from glioma. Am J Clin Pathol. 2004; 121(5): Lassman AB, Holland EC. Incorporating molecular tools into clinical trials and treatment for gliomas? Curr Opin Neurol. Dec 2007; 20(6): PMID Thiessen B, Maguire JA, McNeil K, et al. Loss of heterozygosity for loci on chromosome arms 1p and 10q in oligodendroglial tumors: relationship to outcome and chemosensitivity. J Neuro- Oncol. 2003; 64(3): Spechler SJ, Sharma P, Souza RF, et al. American Gastroenterological Association medical position statement on the management of Barrett's esophagus. Gastroenterology. 2011; 140(3): PMID Yantiss RK. Diagnostic challenges in the pathologic evaluation of Barrett esophagus. Arch Pathol Lab Med. 2010; 134(11): PMID Khara HS, Jackson SA, Nair S, et al. Assessment of Mutational Load in Biopsy Tissue Provides Additional Information About Genomic Instability to Histological Classifications of Barrett's Esophagus. J Gastrointest Cancer. Jan PMID Spechler SJ, Sharma P, Souza RF, et al. American Gastroenterological Association technical review on the management of Barrett's esophagus. Gastroenterology. Mar 2011; 140(3):e18-52; quiz e13. PMID National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology: pancreatic adenocarcinoma, version (discussion update in progress). Available online at: Last accessed June National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology: central nervous system cancers, version Available online at: Last accessed June National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology: esophageal and esophagogastric junction cancers (excluding the proximal 5 cm of the stomach), version Available online at: Last accessed June National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology: hepatobiliary cancers, version Available online at: Last accessed June 2015.

14 Subject: PathFinderTG Molecular Testing Page: 14 of 15 Policy History Date Action Reason June 2012 New Policy September 2013 Update Policy Policy updated with literature search; references 4-13, 16 and 17 added. Policy statement unchanged. September 2014 September 2015 Keywords RedPath Pathfinder Cytology Glioma K-RAS Mutation Loss of Heterozygosity Pancreatic Cysts Update Policy Update Policy Policy updated with literature review; References 2-4, 19-23, added. Barrett esophagus added to policy statement, which is otherwise unchanged. Policy updated with literature review; references 5, 23, and added; reference 21 deleted. The policy statement was revised from not medically necessary to investigational as a correction to align with FDA regulatory status. This policy was approved by the FEP Pharmacy and Medical Policy Committee on September 18, 2015 and is effective October 15, Signature on file Deborah M. Smith, MD, MPH Appendix Appendix Table 1. Categories of Genetic Testing Addressed in Policy No. Category Addressed Yes No 1. Testing of an affected individual s germline to benefit the individual 1a. Diagnostic X 1b. Prognostic X 1c. Therapeutic X 2. Testing cancer cells from an affected individual to benefit the individual a,b,c 2a. Diagnostic X 2b. Prognostic X

15 Subject: PathFinderTG Molecular Testing Page: 15 of 15 2c. Therapeutic X 3. Testing an asymptomatic individual to determine future risk of disease X 4. Testing of an affected individual s germline to benefit family members X 5. Reproductive testing 5a. Carrier testing: preconception X 5b. Carrier testing: prenatal X 5c. In utero testing: aneuploidy X 5d. In utero testing: mutations X 5e. In utero testing: other X 5f. Preimplantation testing with in vitro fertilization X a PathFinderTG Pancreas tests for gene mutations in pancreatic cyst fluid and tissue (potentially precancerous cells). b PathFinderTG Glioma tests for gene mutations in patients with suspected glioma. c PathFinderTG Barrett tests for gene mutations in dysplastic esophageal cells (potentially precancerous cells) in patients with Barrett esophagus.