National Medical Policy

National Medical Policy Subject: Circulating Tumor Cells Policy Number: NMP 513 Effective Date*: June 2012 Updated: June 2017 This National Medical Policy is subject to the terms in the IMPORTANT NOTICE at the end of this document For Medicaid Plans: Please refer to the appropriate State s Medicaid manual(s), publication(s), citation(s), and documented guidance for coverage criteria and benefit guidelines prior to applying Health Net Medical Policies The Centers for Medicare & Medicaid Services (CMS) For Medicare Advantage members please refer to the following for coverage guidelines first: Use Source Reference/Website Link National Coverage Determination (NCD) National Coverage Manual Citation X Local Coverage Determination (LCD)* Circulating Tumor Cell Marker Assays: http://www.cms.gov/medicare-coveragedatabase/search/advanced-search.aspx Article (Local)* X Other MLN Matters. Number: MM8162. Healthcare Common Procedure Coding System (HCPCS) Codes Subject to and Excluded from Clinical Laboratory Improvement Amendments (CLIA) Edits. April 1, 2013: http://www.cms.gov/outreach-and- Education/Medicare-Learning-Network- MLN/MLNMattersArticles/Downloads/MM8162.pd f None Use Health Net Policy Instructions Medicare NCDs and National Coverage Manuals apply to ALL Medicare members in ALL regions. Medicare LCDs and Articles apply to members in specific regions. To access your specific region, select the link provided under Reference/Website and follow the search instructions. Enter the topic and your specific state to find the coverage determinations for your region. *Note: Health Net must follow local coverage determinations (LCDs) of Medicare Administration Contractors (MACs) located outside their Circulating Tumor Cells Jun 17 1

service area when those MACs have exclusive coverage of an item or service. (CMS Manual Chapter 4 Section 90.2) If more than one source is checked, you need to access all sources as, on occasion, an LCD or article contains additional coverage information than contained in the NCD or National Coverage Manual. If there is no NCD, National Coverage Manual or region specific LCD/Article, follow the Health Net Hierarchy of Medical Resources for guidance. Current Policy Statement Health Net, Inc. considers circulating tumor cells investigational for any indication. Although it may help to predict disease progression and survival in some patients with metastatic cancer, there are no conclusive data in the published peer-reviewed medical literature to indicate that this testing can be used to alter the therapy that is offered to patients and improve outcomes. In addition, currently available studies indicate that the sensitivity of circulating tumor cells is relatively low compared with conventional imaging techniques. The current trials have not demonstrated that this testing is equal to or better than any existing tumor markers in its efficacy and clinical utility. Although there are a number of ongoing Clinical Trials, the results will not be posted until completion. An appropriate clinical role for CTC assays remains to be established. Key Words Definitions CTC Circulating tumor cells PFS Progression-free survival. PFS notes the chances of staying free of disease progression for a group of individuals suffering from a cancer after a particular treatment. It is the percentage of individuals in the group whose disease is likely to remain stable (and not show signs of progression) after a specified duration of time. Progression-free survival rates are an indication of how effective a particular treatment is. EpCam Epithelial cell adhesion molecule ECOG The Eastern Cooperative Oncology Group. ECOG is one of the largest clinical cancer research organizations in the U. S. and conducts clinical trials CIs Confidence Intervals. A CI is an interval estimate of a population parameter and is used to indicate the reliability of an estimate. OS Overall survival NoMi Nodal micrometastasis DAPI 4-6-diamidino-2-phenylindole PE Phycoerythrin CK Cytokeratin APC Allophycocyanin HR Hazard ratio. Hazard ratios (HRs) are used to assess the probability of overall survival (OS) and progression free survival (PFS). Circulating Tumor Cells Jun 17 2

MBC PCR RT-PCR qrt-pcr DTCs GC NK OS ROC IDI TFR BM Metastatic breast cancer Polymerase chain reaction. PCR is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Reverse transcription polymerase chain reaction. RT-PCR is a variant of PCR. It is a laboratory technique commonly used in molecular biology where a RNA strand is reverse transcribed into its DNA complement (complementary DNA, or cdna) using the enzyme reverse transcriptase, and the resulting cdna is amplified using PCR. Quantitative real time polymerase chain reaction. qrt-pcr is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. Disseminated tumor cells Gastric cancer Natural killer Overall survival Receiver operator characteristic Integrated discrimination improvement Time to first recurrence Bone marrow Codes Related To This Policy NOTE: The codes listed in this policy are for reference purposes only. Listing of a code in this policy does not imply that the service described by this code is a covered or noncovered health service. Coverage is determined by the benefit documents and medical necessity criteria. This list of codes may not be all inclusive. On October 1, 2015, the ICD-9 code sets used to report medical diagnoses and inpatient procedures have been replaced by ICD-10 code sets. ICD-9 Codes 153.0-153.9 Malignant neoplasm of hepatic flexure malignant neoplasm of colon unspecified site 154.0-154.3 Malignant neoplasm of rectosigmoid junction malignant neoplasm of anus unspecified site 154.8 Malignant neoplasm of other sites of rectum rectosigmoid junction and anus 174.0-174.6 Malignant neoplasm of nipple and areola of female breast malignant neoplasm of axillary tail of female breast 174.8 Malignant neoplasm of other specified sites of female breast 174.9 Malignant neoplasm of breast (female) unspecified site 175.0-175.9 Malignant neoplasm of nipple and areola of male breast Malignant neoplasm of other and unspecified sites of male breast 185 Malignant neoplasm of prostate 196.0 Secondary and unspecified malignant neoplasm of lymph nodes of head, face and neck 198.3 Secondary malignant neoplasm of brain and spinal cord 198.5 Secondary malignant neoplasm of bone and bone marrow ICD-10 Codes C18.2-C18.8 C21.1-C21.8 Malignant neoplasm of colon Malignant neoplasm of anus and anal canal Circulating Tumor Cells Jun 17 3

C77.0-C77.8 Secondary and unspecified malignant neoplasm of lymph nodes C79.11 C79.40-C79.49 Secondary malignant neoplasm of bladder Secondary malignant neoplasm of other parts of nervous system C79.61-C79.62 Secondary malignant neoplasm of ovary (Right and Left) C79.81 Secondary malignant neoplasm of breast CPT Codes 86152 Cell enumeration using immunologic selection and identification in fluid specimen (eg, circulating tumor cells in blood) 86153 Cell enumeration using immunologic selection and identification in fluid specimen (eg, circulating tumor cells in blood); physician interpretation and report, when required HCPCS Codes N/A Scientific Rationale Update June 2017 The Version 2.2017 NCCN guidelines on breast cancer and neuroendocrine cancer have not changed since 2016. There continues to be no information in the 2017 NCCN guidelines on circulating tumor cells for colon, prostate, hepatobiliary, gastric, small cell lung, melanoma, non-small cell lung, or bladder cancer. Scientific Rationale Update June 2016 The NCCN Clinical Practice Guidelines on Breast Cancer (Version 2.2016) notes: The clinical use of circulating tumor cells (CTC) in metastatic breast cancer is not yet included in these guidelines for disease assessment and monitoring. Patients with persistently increased CTC after 3 weeks of first-line chemotherapy have a poor progression free survival (PFS) and overall survival (OS). In spite of its prognostic ability, CTC count has failed to show a predictive value. The NCCN Clinical Practice Guidelines on Neuroendocrine Cancer (Version 1.2016) notes: Circulating tumor cells (CTC) 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 presence of > 1 CTC in 7.5 ml of blood was independently associated with worse progression-free survival (PFS) and overall survival (OS) in patients with varying pre-treated metastatic neuroendocrine tumors from varying primary sites, per a study by Kim et al. (2013). There is no mention of circulating tumor cells in the NCCN Clinical Practice Guidelines on Colon Cancer (Version 2.2016), in the (Version 2.2016) on Prostate Cancer, in the (Version 2.2016) on Gastric Cancer, in the (Version 2.2016) on Bladder Cancer, in the (Version 2.2016) on Melanoma, in the (Version 2.2016) on Hepatobiliary cancers, in the (Version 2.2015) on Ovarian Cancer, in the (Version 4.2016) on NSCLC, and in the (Version 1.2016) on Small Cell Lung Cancer. Guidelines from the American Society for Clinical Oncology (2016) state: "The clinician should not use circulating tumor cells to guide decisions on adjuvant systemic therapy." (i.e., Type: evidence based. Evidence quality: intermediate. Strength of recommendation). Lv et al. (2015) completed a meta-analysis based on a comprehensive search of articles that focused on metastatic breast cancer (MBC) patients and detected circulating tumor cells (CTCs) using the CellSearch system. The associations between Circulating Tumor Cells Jun 17 4

CTCs and survival rate and clinicopathological parameters, including molecular pattern, metastatic region and treatment response, were evaluated. This metaanalysis included 24 studies (3701 MBC patients), 13 prospective studies and 11 retrospective studies. The authors found that CTCs were more frequently detected with HER2 + primary tumors (pooled RR = 0.73, 95% CI = 0.63-0.84). Additionally, higher CTC numbers indicated a worse treatment response (RR = 0.56, 95 % CI = 0.40-0.79), poorer PFS (RR = 0.64, 95% CI = 0.56-0.73) and poorer OS (RR = 0.69, 95 % CI = 0.64-0.75) in MBC patients. Based on these results, the authors propose that HER2 positivity could be a significant risk factor for the presence of CTCs. Additionally, CTCs have a significant prognostic value for MBC patients. Therefore, the authors feel that CTCs should be continually monitored to guide the treatment of MBC patients, especially those with HER2 + primary tumors. Paoletti et al. (2015) completed a randomized Phase II Clinical Trial to determine the significance of circulating tumor cells (CTC) and whether or not they are prognostic in metastatic breast cancer (MBC). The authors tested whether EpCAM-based capture system (CellSearch) is effective in patients with triple-negative (TN) MBC, and whether CTC apoptosis and clustering enhances the prognostic role of CTC. CTC enumeration and apoptosis were determined using the CXC CellSearch kit at baseline and days 15 and 29 in blood drawn from TN MBC patients of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) with or without tigatuzumab. Association between levels of CTC and patient outcomes was assessed using logistic regression, Kaplan-Meier curves, and Cox proportional hazards modeling. Nineteen of 52 (36.5%), 14 of 52 (26.9%), and 13 of 49 (26.5%) patients who were evaluable had elevated CTC ( 5 CTC/7.5 ml whole blood) at baseline and at days 15 and 29, respectively. Patients with elevated versus not elevated CTC at each time point had worse progression-free survival (PFS; P = 0.005, 0.0003, 0.0002, respectively). The odds of clinical benefit response for those who had elevated versus low CTC at baseline and days 15 and 29 were 0.25 (95% CI: 0.08-0.84; P = 0.024), 0.19 (95% CI: 0.05-0.17; P = 0.014), and 0.06 (95% CI: 0.01-0.33; P = 0.001), respectively. There was no apparent prognostic effect comparing CTC apoptosis versus nonapoptosis. Presence of CTC cluster at day 15 and day 29 was associated with shorter PFS. CTC were detected using CellSearch assay in approximately one-third of TN MBC patients. Elevated CTC at baseline and days 15 and 29 were prognostic, and reductions in CTC levels reflected response. Helissey et al. (2015) The clinical validity of circulating tumor cell (CTC) count changes during chemotherapy in metastatic breast cancer patients has been validated, but its clinical utility remains to be demonstrated. The authors report the non-randomized run-in phase of the CirCe01 trial which was designed to evaluate CTC changes and thresholds to other palliative prognostic scores and establish CTC thresholds to be used in the randomized part of the study. CTC count (CellSearch) and other prognostic parameters (serum albumin level, lymphocyte level, LDH level, prognostic inflammatory and nutritional index (PINI) and Barbot's score) were assessed in 56 metastatic breast cancer patients before the first cycle of third line chemotherapy. Early changes of CTC count were correlated with treatment outcome. Independent prognostic markers in multivariate analysis were: low serum albumin (HR=11.1), poor performance status (HR=3.8), 5CTC/7.5ml (HR=3.8) and triple negative subtype (HER2+ and hormone positive vs triple negative: both HR=0.2). Among patients with 5CTC/7.5ml at baseline, a composite criteria (<5CTC/7.5ml or relative decrease -70% of the baseline CTC count) showed better prognostication for PFS (p=0.002). Mego et al. (2015) Circulating tumor cells (CTCs) are an independent prognostic factor for progression-free survival (PFS) and overall survival (OS) in patients with metastatic breast cancer. Inflammatory breast cancer (IBC) is one of the most Circulating Tumor Cells Jun 17 5

aggressive forms of breast cancer. The prognostic value of a CTC count in newly diagnosed IBC has not been established. The aim of this study was to assess the prognostic value of a baseline CTC count in patients with newly diagnosed IBC. This retrospective study included 147 patients with newly diagnosed IBC (77 with locally advanced and 70 with metastatic IBC) treated with neoadjuvant therapy or first-line chemotherapy during the period from January 2004 through December 2012 at The University of Texas MD Anderson Cancer Center. CTCs were detected and enumerated by using the CellSearch system before patients were started with chemotherapy. The proportion of patients with 1 CTC was lower among patients with stage III than among patients with metastatic IBC (54.5% versus 84.3%; P=0.0002); the proportion of patients with 5 CTCs was also lower for stage III than for metastatic IBC (19.5% versus 47.1%; P=0.0004). Patients with fewer than five CTCs had significantly better progression-free survival (PFS) (hazard ratio (HR)=0.60; P=0.02) and overall survival (HR=0.59; P=0.03) than patients with five or more CTCs. Among patients with stage III IBC, there was a nonsignificant difference in PFS (HR=0.66; 95% confidence interval (CI), 0.31 to 1.39; P=0.29) and OS (HR=0.54; 95% CI, 0.24 to 1.26; P=0.48) in patients with no CTCs compared with patients with one or more CTCs. In multivariate analysis, CTC was prognostic for PFS and OS independent of clinical stage. CTCs can be detected in a large proportion of patients with newly diagnosed IBC and are a strong predictor of worse prognosis in patients with newly diagnosed IBC. Magbaunua et al. (2015) Recent developments in rare-cell technology have led to improved blood-based assays that allow for the reliable detection, enumeration, and more recently, genomic profiling of circulating tumor cells (CTC). The authors evaluated two different approaches for enumeration of CTCs in a prospective therapeutic study of patients with metastatic triple-negative breast cancer (TNBC). The CellSearch system, a commercially available and U.S. Food and Drug Administration (FDA)-cleared assay for CTC enumeration, and IE/FC, an alternative method using EPCAM-based immunomagnetic enrichment and flow cytometry that maintains cell viability, were used to enumerate CTCs in the blood of patients with metastatic TNBC. CTC numbers were assessed at baseline and 7 to 14 days after initiation of therapy with cetuximab ± carboplatin in a phase II multicenter clinical trial (TBCRC 001). CTC numbers from two methods were significantly correlated at baseline (r = 0.62) and at 7 to 14 days (r = 0.53). Baseline CTCs showed no association with time-to-progression (TTP), whereas CTCs at 7 to 14 days were significantly correlated with TTP (CellSearch P = 0.02; IE/FC P = 0.03). CTCs at both time points were significantly associated with overall survival (OS) [CellSearch: baseline (P = 0.0001) and 7 to 14 days (P < 0.0001); IE/FC: baseline (P = 0.0009) and 7 to 14 days (P = 0.0086)]. Our findings demonstrate that CTC enumeration by two different assays was highly concordant. In addition, results of both assays were significantly correlated with TTP and OS in patients with TNBC. The IE/FC method is also easily adapted to isolation of pure populations of CTCs for genomic profiling. Scientific Rationale Update June 2015 The NCCN Clinical Practice Guidelines on Breast Cancer (Version 2. 2015) mentions the circulating tumor cells only under the section on Staging. It mentions defining a new category of MO(l+) disease referring to tumor cells detectable in bone marrow or circulating tumor cells or found incidentally in other tissues if not exceeding 0.2 mm. Category MO(l+) refers to a stage with no clinical or radiographic evidence of distant metastasis, but deposits of molecularly or microscopically detected tumor cells in circulating blood, bone marrow or other nonregional nodal tissue that are no larger than 0.2mm in a patient without symptoms or signs of metastasis. There is no mention of circulating tumor cells in the NCCN Clinical Practice Guidelines on Colon Cancer (Version 2.2015), in the Guidelines on Prostate Cancer (Version Circulating Tumor Cells Jun 17 6

1.2015), in the Guidelines on Gastric Cancer (Version 3.2015), in the Guidelines on Hepatobilary Cancer (Version 1.2015), in the Guidelines on Ovarian Cancer (Version 1.2015), in the Guidelines on Melanoma (Version 3.2015), in the Guidelines on Non- Small Cell Lung Cancer (Version 6.2015) or in the Guidelines on Bladder Cancer (Version 1.2015). There are a number of Clinical Trials on circulating tumor cells that are currently recruiting participants. There are also a number that are completed with no study results posted. Wallwiener et al. (2015) completed a retrospective study on metastatic breast cancer (MBC) patients starting a new line of systemic treatment, and were eligible for the study. HER2 status of circulating tumor cells (CTCs) was determined by CellSearch. HER2 status of primary (PRIM) and metastatic (MET) tumor tissue was determined by immunohistochemistry. Data were analyzed using Kaplan-Meier plots. One hundred seven patients (median age (range) 57 (33-81) years) were included. 100/107 (93 %) patients were followed-up for a median [95 % confidence interval (CI)] of 28.5 [25.1-40.1] months. Of 37/107 (35 %) CTC-HER2-positive patients only 10 (27 %) were PRIM-HER2-positive. 6/46 (13 %) patients were MET-HER2- positive; only 2/10 (20 %) CTC-HER2-positive patients were MET-HER2-positive. Overall accuracy between CTC-HER2 expression and PRIM-HER2 and MET-HER2 status was 69 % and 74 %, respectively. Kaplan-Meier plots of PFS and OS by CTC- HER2 status revealed significantly longer median [95 % CI] PFS of CTC-HER2- positive versus CTC-HER2-negative patients (7.4 [4.7-13.7] versus 4.34 [3.5-5.9] months; p=0.035). CTC-HER2-positive status showed no significant difference for OS (13.7 [7.7-30.0] versus 8.7 [5.9-15.3] months; p=0.287). HER2 status can change during the course of breast cancer. CTC phenotyping may serve as an easy-toperform "liquid biopsy" to reevaluate HER2 status and potentially guide treatment decisions. Further, prospective studies are needed. Chudasama et al. (2015) completed a study on nine individuals with primary or metastatic lung tumors who underwent RFA therapy from June to November 2013. Blood samples were taken before and after RFA, and filtered through the ScreenCell circulating tumor cell (CTC) capture device. ScreenCell is a non-invasive technology for isolating CTCs from whole blood, and potentially any atypical cell in biological fluids. A general increase in CTCs in 7 out of the 9 cases was found, the largest increases were seen in the metastatic group. This study demonstrates that the manipulation and ablative procedure of lung tumors may lead to immediate dissemination of tumor cells, the effects of which are unknown and require further investigation. There continues to be no conclusive data in the published peer-reviewed medical literature to indicate that this testing can be used to alter the therapy that is offered to cancer patients and improve outcomes. None of the studies have shown that this testing is equal to or better than any existing tumor markers in its efficacy and clinical utility. Scientific Rationale Update June 2014 Huang et al (2014) reported the prognostic significance of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) in patients with gastric cancer (GC) is controversial. The authors performed a meta-analysis to assess its correlation with clinicopathological characteristics and prognostic significance in GC. PubMed, Embase, the Cochrane database, the Science citation index, the CNKI database and the references of relevant studies were systematically searched (up to November, 2013). Using the random-effect model, the meta-analysis was completed with odds ratio (OR), risk ratio, hazard ratio (HR) and 95% confidence intervals (CI) as effect Circulating Tumor Cells Jun 17 7

values. Twenty-six studies containing 2,566 patients with GC were analyzed. The overall analysis showed that the incidence difference of tumor cells (CTCs/DTCs) was significant when comparing the stage I/II group to the stage III/IV group (OR = 0.36, CI [0.23, 0.56]), the Lauren diffuse group to the intestinal group (OR = 2.06, CI [1.06, 4.00]), the poorly differentiated group to the well/moderate group (OR = 1.65, CI [1.10, 2.50]), the lymphatic involvement positive group to the positive group (OR = 2.92, CI [1.00, 8.55]). The detection of CTCs/DTCs was significantly related with the disease-free survival of patients (HR = 3.42, CI [2.39, 4.91]) and the detection of CTCs in peripheral blood was significantly related with the overall survival of patients (HR = 2.13, CI [1.13, 4.03]). The authors concluded the metaanalysis indicates that detection of CTCs/DTCs is associated with prognosis for patients with GC and thus could act as a basis for GC staging. The NCCN guidelines on gastric cancer (2.2013) do not mention the use of CTC s. Santos et al (2014) reported CTC s in metastatic cancer patients have been found to be a promising biomarker to predict overall survival and tumor progression in these patients. A relatively high number of CTCs has been correlated with disease progression and poorer prognosis. They designed a study to assess innate immune system function, known to be responsible for the immune defense against developing neoplasms, in metastatic cancer patients with CTCs. The aim was to provide a link between indication of poorer prognosis, represented by the number of CTCs to the cytotoxic activity of natural killer cells, an important component of the innate immune system, and to represent a promising expanded approach to management of metastatic cancer patients with CTCs. Seventy-four patients, with metastatic breast, colorectal, or prostate cancer, were recruited for this study. Using a flow cytometric assay, we measured natural killer (NK) cell cytotoxicity against K562 target cells; and CTCs were enumerated using the CellSearch System. Toll-like receptors 2 and 4 expression was also determined by flow cytometry. We found that within each of our three metastatic cancer patient groups, NK cell cytotoxic activity was decreased in patients with a relatively high number of CTCs in peripheral blood compared to patients with a relatively low number of CTCs. In the breast and prostate cancer group, patients with CTCs greater than 5 had decreased NK cell cytotoxicity when compared to patients with less than 5 CTCs. In the colorectal cancer group, we found that 3 or more CTCs in the blood was the level at which NK cell cytotoxicity is diminished. Additionally, the authors found that the toll-like receptors 2 and 4 expression was decreased in intensity in all the metastatic cancer patients when compared to the healthy controls. Furthermore, within each cancer group, the expression of both toll-like receptors was decreased in the patients with relatively high number of CTCs, i.e. greater than 5 for the breast and prostate cancer group and greater than 3 for the colorectal cancer group, compared to the patients with relatively low number, i.e. less than 5 or 3, respectively. Treatment options to increase NK cell cytotoxic activity should be considered in patients with relatively high numbers of CTCs. At this time, NCCN guidelines on breast, colon or prostate cancer do not address the use of CTC s in their guidelines. Goldkorn et al (2014) assessed the prognostic value of CTCs for overall survival (OS) and disease response in S0421, a phase III trial of docetaxel plus prednisone with or without atrasentan. CTCs were enumerated at baseline (day 0) and before cycle two (day 21) using CellSearch. Baseline counts and changes in counts from day 0 to 21 were evaluated for association with OS, prostate-specific antigen (PSA), and RECIST response using Cox regression as well as receiver operator characteristic (ROC) curves, integrated discrimination improvement (IDI) analysis, and regression trees. Median day-0 CTC count was five cells per 7.5 ml, and CTCs < versus five per 7.5 ml were significantly associated with baseline PSA, bone pain, liver disease, hemoglobin, alkaline phosphatase, and subsequent PSA and RECIST response. Median OS was 26 months for < five versus 13 months for five CTCs per 7.5 ml at Circulating Tumor Cells Jun 17 8

day 0 (hazard ratio [HR], 2.74 [adjusting for covariates]). ROC curves had higher areas under the curve for day-0 CTCs than for PSA, and IDI analysis showed that adding day-0 CTCs to baseline PSA and other covariates increased predictive accuracy for survival by 8% to 10%. Regression trees yielded new prognostic subgroups, and rising CTC count from day 0 to 21 was associated with shorter OS (HR, 2.55). Investigators concluded the data validates the prognostic utility of CTC enumeration in a large docetaxel-based prospective cohort. Baseline CTC counts were prognostic, and rising CTCs at 3 weeks heralded significantly worse OS, potentially serving as an early metric to help redirect and optimize therapy in this clinical setting. Gazzaniga et al (2014) reported High-risk non-muscle invasive bladder cancer (NMIBC) progresses to metastatic disease in 10-15% of cases, suggesting that micrometastases may be present at first diagnosis. The prediction of risks of progression relies upon EORTC scoring systems, based on clinical and pathological parameters, which do not accurately identify which patients will progress. Aim of the study was to investigate whether the presence of CTC may improve prognostication in a large population of patients with Stage I bladder cancer who were all candidate to conservative surgery. A prospective single center trial was designed to correlate the presence of CTC to local recurrence and progression of disease in high-risk T1G3 bladder cancer. One hundred two patients were found eligible, all candidate to transurethral resection of the tumor followed by endovesical adjuvant immunotherapy with BCG. Median follow-up was 24.3 months (minimum-maximum: 4-36). The FDA-approved CellSearch System was used to enumerate CTC. Kaplan- Meier methods, log-rank test and multivariable Cox proportional hazard analysis was applied to establish the association of circulating tumor cells with time to first recurrence (TFR) and progression-free survival. CTC were detected in 20% of patients and predicted both decreased TFR (log-rank p < 0.001; multivariable adjusted hazard ratio [HR] 2.92 [95% confidence interval: 1.38-6.18], p = 0.005), and time to progression (log-rank p < 0.001; HR 7.17 [1.89-27.21], p = 0.004). The authors concluded the findings provide evidence that CTC analyses can identify patients with Stage I bladder cancer who have already a systemic disease at diagnosis and might, therefore, potentially benefit from systemic treatment. Seeberg et al (2014) assessed the prognostic and predictive value of CTC s and DTCs in bone marrow (BM) in patients with colorectal liver metastasis referred to surgery. A total of 194 patients were included. Treatment of the patients was decided in a multidisciplinary team. BM aspirates and blood samples were collected at surgery, or in local anesthesia in nonresectable patients. CTCs were disclosed with CellSearch System, DTC with immunocytology. Liver resection was completed in 153 patients. Forty-one patients were nonresectable, 22 preoperatively and 19 intraoperatively. The median follow-up was 22 (range 1-61) months. Relapse was diagnosed in 103 of the resected patients. Totally, 67 patients died of cancer. CTCs were detected in 19.6% of the patients. CTC positivity was significantly higher in nonresectable (46%) than in resectable patients (11.7%), P < 0.001. 13.8% of the patients had 2 or more CTCs, 31% of the nonresectable and 9.1% of the resectable patients (P = 0.001). Patients with 2 or more CTCs experienced reduced time to relapse/progression, both analyzing all patients (P = 0.002) and analyzing resectable patients (P < 0.001). Two or more CTCs was a strong predictor of progression and mortality in all subgroups of patients, together with more than 3 liver metastases, R1 resection, and extrahepatic disease. DTCs were detected in 9.9% of the patients, but not associated with clinical outcome in resectable patients. The authors concluded CTCs predict nonresectability and impaired survival. CTC analysis should be considered as a tool for decisionmaking before liver resection in these patients. Circulating Tumor Cells Jun 17 9

NCCN guidelines on meuroendocrine tumors (2.2014) states: Circulating tumor cells (CTC) have 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 study found that the presence of > 21 CTC in 7.5 ml of blood was independently associated with worse progression free survival and overall survival in patients with varyingly pre-treated metastatic neuroendocrine tumors from various primary sites. More research is required before these and other new molecular assays are routinely used. Scientific Rationale Update June 2013 Andreopoulou et al. (2012) The detection of CTCs prior to and during therapy is an independent and strong prognostic marker, and it is predictive of poor treatment outcome. A major challenge is that different technologies are available for isolation and characterization of CTCs in peripheral blood (PB). The authors compare the CellSearch system and AdnaTest BreastCancer Select/Detect, to evaluate the extent that these assays differ in their ability to detect CTCs in the PB of MBC patients. CTCs in 7.5 ml of PB were isolated and enumerated using the CellSearch, before new treatment. Two cutoff values of 2 and 5 CTCs/7.5 ml were used. AdnaTest requires 5 ml of PB to detect gene transcripts of tumor markers (GA733-2, MUC-1, and HER2) by RT-PCR. AdnaTest was scored positive if 1 of the transcript PCR products for the 3 markers were detected at a concentration 0.15 ng/μl. A total of 55 MBC patients were enrolled. 26 (47%) patients were positive for CTCs by the CellSearch ( 2 cutoff), while 20 (36%) were positive ( 5 cutoff). AdnaTest was positive in 29 (53%) with the individual markers being positive in 18% (GA733-2), 44% (MUC-1), and 35% (HER2). Overall positive agreement was 73% for CTC 2 and 69% for CTC 5. These preliminary data suggest that the AdnaTest has equivalent sensitivity to that of the CellSearch system in detecting 2 or more CTCs. While there is concordance between these 2 methods, the AdnaTest complements the CellSearch system by improving the overall CTC detection rate and permitting the assessment of genomic markers in CTCs. Banys et al. (2012) The detection of disseminated tumor cells in bone marrow is a common phenomenon seen in 30-40% of primary breast cancer patients. The presence of disseminated tumor cells at diagnosis as well as the persistence of disseminated tumor cells is strongly associated with poor clinical outcome. Since bone marrow biopsies are not well tolerated by many patients, the evaluation of circulating tumor cells in the blood might become a desired alternative. Circulating tumor cells are routinely detected, depending on stage of the disease and methodology, in 10-80% of breast cancer patients. Recent studies have shown a prognostic potential of circulating tumor cells in both primary and metastatic settings. The evaluation of circulating tumor cells may become one of the crucial markers for prediction of survival and therapy monitoring, and its characterization might enable specific targeting of minimal residual, and metastatic disease. Pierga et al. (2012) Circulating tumor cells (CTCs) are a prognostic marker in metastatic breast cancer, but comparisons with serum tumor markers (CA 15-3, carcinoembryonic antigen and lactate dehydrogenase) variations are needed. CTCs were counted with CellSearch at baseline, before cycle 2 (C2) and cycle 3 or 4 (C3/4) in 267 metastatic breast cancer patients on first-line chemotherapy with/without targeted therapy. Baseline CTC detection rate was 65% with 1 CTC/7.5 ml threshold and 44% with 5 CTC/7.5 ml and was independent of subtypes (luminal, triple negative, human epithelial growth factor receptor 2 (HER2)+). CTCs were associated with tumor markers, bone/liver involvement, tumor burden and performance status. CTC detection 1 CTC/7.5 ml was a strong prognostic factor for progression-free survival (PFS), P < 0.0001. Threshold of CTC 5 was statistically significant for PFS and overall survival (OS), P = 0.03 on multivariate analysis. Circulating Tumor Cells Jun 17 10

Among patients with 5 CTC/7.5 ml at baseline, 50% had <5 CTC/7.5 ml at C2. Changes were correlated with both PFS and OS (P < 0.0001). All patients receiving anti-her2 therapy had <5 CTC/7.5 ml after three cycles of treatment. This is the largest prospective series validating the prognostic value of CTC independently from serum tumor marker. Elevated CTCs before C2 are an early predictive marker of poor PFS and OS, which could be used to monitor treatment benefit. CTC decrease under treatment seems stronger with targeted therapy. There continues to be no information in NCCN (2013) for Prostate Cancer or Colon Cancer regarding CellSearch or circulating tumor cells. The NCCN 2013 guidelines on Breast Cancer do mention circulating cells. However, this is the same verbiage from NCCN that is noted in the 'Initial Scientific Rationale'. NCCN has no additional information on CellSearch or circulating tumor cells. Clinical Trials: Two Clinical Trials noted within the initial scientific rationale have not been updated since 2011. They are: ClinicalTrials.gov Identifier number is NCT01349842, has not been updated since May 6, 2011 Clinicaltrial.gov. Identifier number NCT01116869, has not been updated since September 26, 2011. A Phase 2, interventional, Clinical Trial on 'Purged Circulating Tumor Cells (CTCs) From Metastatic Breast Cancer' has been completed. The ClinicalTrials.gov Identifier is NCT00429182, and it was last updated on February 28, 2013. The recruitment period was February 27, 2007 to July 25, 2011. All recruitment was done at The University of Texas (UT) MD Anderson Cancer Center. The goal of this clinical research study is to learn the relationship of high-dose chemotherapy (HDCT) and circulating tumor cells (CTCs) in controlling metastatic breast cancer. The study also will investigate the role of CTCs in breast cancer. 32 patients started the study, but only 25 completed it, with the median age of 44 with ages from 29-59 included. 21 members had high dose chemotherapy with purged autologous hematopoietic stem cell transplantation (AHST). The number of circulating cells were measured at 1 month post AHST. Out of the 21 participants, 9 had a reduction of CTCs post treatment. No statistical analysis was provided for the number of participants with reduction in CTCs following High-dose Chemotherapy with purged autologous stem cell products. 26 participants were analyzed, and 10.6 had a median progression free survival (PFS) of 1-16.4 months. Serious adverse events from the high dose chemotherapy affected 2/32 participants. Thrombocytopenia affected 1/32 participants. 1/32 participants died. Other adverse reactions, such as gastrointestinal disorders, affected 27/32 participants. Limitations of the study, such as early termination leading to small numbers of participants analyzed, and technical problems with measurement leading to unreliable or uninterpretable data, were noted. Another Clinical Trial on Identifying Circulating Breast Cancer Cells in Women With Metastatic Breast Cancer, with the Identifier number of NCT00897338, was also noted in the initial scientific rationale, however, was last updated in October 2012. There is no estimated completion date. A randomized Clinical Trial on ' Medico-economic Interest of Taking Into Account Circulating Tumor Cells (CTC) to Determine the Kind of First Line Treatment for Metastatic, Hormone-receptors Positive, Breast Cancers' is now recruiting Circulating Tumor Cells Jun 17 11

participants. The ClinicalTrials.gov Identifier is NCT01710605, and it was last updated on October 18, 2012. The purpose of this study is to evaluate the medicoeconomic interest of taking into account circulating tumor cells (CTC) to determine the kind of first line treatment for metastatic, hormone-receptors positive, breast cancers. In the standard arm, the kind of treatment will be decided by clinicians, taking into account the criteria usually used in this setting. In the CTC arm, the type of treatment will be decided by CTC count: hormone-therapy if <5CTC/7.5mll (CellSearch technique) or chemotherapy if =5. The main medical objective is to demonstrate the non-inferiority of the CTC-based strategy for the progression-free survival: 994 patients are needed, and will be accrued in French cancer centers. Secondary clinical objectives are to compare toxicity, quality of life and overall survival between the two arms. The medico-economic study will compare cost per progression-free life years gained of the two strategies. The financial impact of centralized (one platform) vs decentralized (several platforms) CTC testing will be evaluated. The estimated primary completion date is March 2016. Another Clinical Trial on ' Circulating Tumor Cells in Lung Cancer (CIRCUBRONCH)' is also recruiting participants. The ClinicalTrials.gov Identifier is NCT01658332, and it was last updated on August 6, 2012. This study is a prospective, monocentrique trial analyzing the identification of circulating tumor cells in stage IIIB, and IV non-small cell lung cancers. Circulating tumor cells identification is a new field of research in oncology, and some studies have been conducted with success on breast and prostate cancer. Nearly 80% of lung cancers are diagnosed in an advanced stage (IIIB, and IV), and this is the primary objective for circulating cells identification and monitoring. The secondary objective is the predictive value of the monitoring of circulating tumor cells on the therapeutic response. Prognostic value of identification of circulating tumor cells at the time of diagnosis. The procedure consists of the detection of circulating tumor cells with CellSearch system (Veridex), and a cut-off of 5 cells/7.5 ml of blood, with 200 patients. The statistical analysis is trying to prove that detection of circulating tumor cells is predicted in approximately 20% of stage IIIB, and IV non-small cell lung cancers included in this study. The cut-off is 5 circulating tumor cells per 7.5 ml of blood. A Clinical Trial on ' Detection and Enumeration of Circulating Tumor Cells in Rectal Cancer' is also recruiting participants. TheClinicalTrials.gov Identifier is NCT01671891, and it was last updated on August 21, 2012. Rectal cancer is one of the most common malignant tumors in the world. However, there's also no reliable and sensitive method to monitor diseases and evaluate therapy responses till now. Circulating tumor cells, which could reflect tumor's status correctly and reliably, may be a promising method in this field. This study is to investigate the role of circulating tumor cells in evaluating and predicting the responses of chemoradiotherapy in rectal cancer. The estimated study completion date is February 2013, however, it is now May 2013, and this study is still recruiting participants. Another Clinical Trial on ' Culture and Characterization of Circulating Tumor Cells (CTC) in Melanoma and Other Cancers' is also recruiting participants. The ClinicalTrials.gov Identifier is NCT01528774, and was last updated on April 26, 2013. The purpose of this study is to determine if circulating tumor cells (CTC) can be accurately detected and isolated from the blood of participants with melanoma using novel laboratory techniques. Blood samples will be collected from participants with melanoma, and also from participants with other solid tumor cancers and healthy volunteers for purposes of comparison. Relevant information will be collected from participant's medical record and stored in a coded manner in a password-protected format. This information will be used to look for correlations of research results on blood samples to participant's medical condition. Test results will not be given to Circulating Tumor Cells Jun 17 12

participants or their physicians. In some cases, CTC may be grown for long-term cell lines for further research. The estimated primary completion date is February 2014. A Clinical Trial on 'Cancer Stem Cell Markers and Prognostic Markers in Circulating Tumor Cells' is recruiting participants. The ClinicalTrials.gov Identifier is NCT01286883, and was last updated on April 11, 2013. The study will enroll patients with metastatic colorectal cancer receiving chemotherapy. A total of approximately 22 cc of blood will be drawn during various chemotherapy infusions. Additional proposed laboratory studies may unravel important biological insights into the relationship of circulating tumor cell genomic and genetic profiles as they compare to the primary tumors. Additionally the investigators hope to gain an understanding of potential subgroups of patients that have very high numbers of circulating tumor cells or those with early relapse of circulating tumor cells after early reduction of circulating tumor cell numbers. The estimated primary completion date is December 2014. Another Clinical Trial on 'Enumeration and Molecular Characterization of Circulating Tumour Cells in Women With Metastatic Breast Cancer' is also recruiting participants. The ClinicalTrials.gov Identifier is NCT01322893, and it was last updated on April 19, 2013. Women with metastatic breast cancer, receiving first line treatment of any kind (chemotherapy, endocrine treatment or treatment with antibodies) will be included in this trial. In connection to treatment, blood samples for determination and enumeration of circulating tumour cells will be collected at different time points. Serum and plasma will be collected and stored for future analysis of RNA and DNA. The estimated primary completion date is June 2013. A Clinical Trial on 'Regional Anesthesia Versus General Anesthesia on Circulating Tumor Cells (CTC)' is recruiting participants. The ClinicalTrials.gov Identifier is NCT01716065, and it was last updated on November 14, 2012. The purpose of the study is to determine whether the type of anesthesia during breast cancer surgery has any impact on the way a patient's immune system functions for a brief period after surgery. If the investigators find that one type of anesthesia versus the other is more beneficial to a patient's immune system, then the investigators may use this information to design a larger study to exam the effect of anesthesia better. The completion date is not noted. Another Clinical Trial on ' Study of Circulating Tumor Cells Before and After Treatment in Patients With Metastatic Melanoma' is recruiting participants. The ClinicalTrials.gov Identifier is NCT01573494, and it was last updated on August 6, 2012. The estimated primary completion date is May 2014, with the primary objective to determine the effect of treatment on the number of circulating melanoma cells in patients with metastatic melanoma. Scientific Rationale Initial Circulating tumor cells (CTCs) are extremely rare in healthy individuals and patients with nonmalignant diseases but are present in patients with various carcinomas with a wide range of frequencies. CTCs are malignant cells that are found in the peripheral blood and originate from primary or metastatic tumors. Detection of elevated CTCs during therapy is being proposed as an indication of subsequent rapid disease progression and mortality in breast, colorectal and prostate cancer. CTCs represent the point in the metastatic process of solid tumors when cells from a primary tumor invade, detach, disseminate, colonize and proliferate in a distant site. The majority of the data comes from studies of women with metastatic breast cancer. CTCs have also been investigated as an additional prognostic factor in Circulating Tumor Cells Jun 17 13

nonmetastatic breast cancer and are proposed to determine the need for additional adjuvant chemotherapy. Research has focused on the detection and development of methods with increased sensitivity and specificity to identify circulating tumor cells (CTCs). There are several methods of detecting CTCs which are in various stages of research and development. CellSearch System One of these methods is the CellSearch System (Veridex), which is a FDA approved in vitro diagnostic device for serial monitoring of metastatic disease, and for detecting CTCs in the whole blood. Genoptix is a medical Laboratory that uses the FDA-cleared CellSearch Circulating Tumor Cell (CTC) Test for metastatic breast, prostate, and colorectal cancers. The test is an aid in monitoring the therapy of patients with these metastatic cancers. CTC testing is an adjunct to radiology in metastatic breast cancer and to prostatespecific antigen (PSA) testing in metastatic prostate cancer. CellSearch is an example of circulating tumor cells. It has received U.S. Food and Drug Administration (FDA) marketing clearance through the 510(k) process for monitoring metastatic breast cancer (January 2004), for monitoring metastatic colorectal cancer (November 2007), and for monitoring metastatic prostate cancer (February 2008). It uses automated instruments manufactured by Immunicon Corporation for sample preparation (Cell Tracks AutoPrep) and analysis (CellSpotterAnalyzer). The CellSearch System uses immunofluorescent technology. This involves identification of the circulating tumor cells in the blood, which are tagged using antibody coated magnetic beads that recognize cell surface antigens. The cells are then labeled with fluorescent dyes, which can then be quantified by a semiautomated fluorescent-based microscopy system. This automated immunomagnetic selection of circulating tumor cells (CTCs) is based on an epithelial cell adhesion molecule (EpCAM), known as an anti-epcam antibody cell capture. To perform this assay, a 7.5 ml. of blood is incubated with EpCAM antibody covered ferroparticles (nanotechnology). Circulating epithelial cells that express EpCAM are isolated in a magnetic field. The CellSearch system uses 3 stains to help distinguish epithelial cells from contaminating leukocytes and nonspecific debris: 4-6-diamidino-2-phenylindole (DAPI) stains the nuclei of cells and helps identify viable cells, phycoerythrin (PE)- labeled cytokeratin (CK) antibodies (CK 8, 18, and 19) recognize epithelial cells, allophycocyanin (APC)-labeled CD45 antibodies identify contaminating leukocytes. Antibodies used in the CellSearch assay are targeted at cell markers (EpCAM and cytokeratins 8, 18, and 19) expressed by adenocarcinomas. CTCs that do not express these markers will not be detected by the CellSearch assay, whereas CTCs from non-breast malignancies expressing these markers may be detected. CellSearch test results should be interpreted in conjunction with other clinical and laboratory findings. The resulting epithelial-enriched fluid is then placed in a cell presentation device (MagNest) that attracts the magnetically labeled epithelial cells to the surface of the cartridge. The cartridge is placed on the CellTracks Analyzer, a fluorescence-based microscopy system that scans the surface of the cartridge to acquire cell images to visualize DAPI-labeled nuclei, PE-labeled CK, and APC-labeled CD45. Circulating Tumor Cells Jun 17 14

The assay findings are verified by a pathologist and issued in a report; a CTC count of greater than >5 cells per 7.5 ml of whole blood prior to therapy or at first followup is predictive of shorter progression free survival (PFS) and shorter overall survival (OS). A decrease in the number of CTCs to less than <5cells per 7.5 ml of whole blood from baseline to first follow-up is associated with longer progression free survival (PFS) and longer overall survival (OS). In addition, it has been noted that more than 5 cells per 7.5 ml of whole blood predicts worse prognosis in patients with known recurrent breast and prostate cancer, and more than 3 cells are predictive of shorter progression free survival (PFS) and overall survival (OS) in metastatic colorectal cancer. Studies Bidard et al. (2010) reported on clinical outcomes of 115 nonmetastatic breast cancer patients according to CTC detection. At baseline, 23% of patients were CTC positive, but only 10% had >1 CTC/7.5 ml of blood. After a median follow-up of 36 months, CTC detection before chemotherapy was an independent prognostic factor for both distant metastasis-free survival (DMFS) (p=0.01, relative risk [RR]=5.0, 95% confidence interval [CI]) 1.4 17) and overall survival (OS) (p=0.007, RR=9, 95% CI 1.8 45). The detection of CTC after chemotherapy was found to be of less significance (p=0.07 and 0.09, respectively). The authors conclude that detection of 1 CTC/7.5 ml before neoadjuvant chemotherapy can accurately predict overall survival. Further trials are needed to confirm the results. Van der Auwera et al. (2010) The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardization of technology hampers the implementation of CTC measurement in clinical routine practice. This study was designed to directly compare three techniques for detecting CTCs in blood samples taken from 76 patients with metastatic breast cancer (MBC) and from 20 healthy controls: the CellSearch CTC System, the AdnaTest Breast Cancer Select/Detect and a previously developed real-time quantitative real time polymerase chain reaction (qrt-pcr) assay for the detection of cytokeratin (CK-19) and mammaglobin transcripts. As a result, 36% of patients with MBC were positive by the CellSearch System, 22% by the AdnaTest, 26% using RT PCR for CK-19 and 54% using RT PCR for mammaglobin. Samples were significantly more likely to be positive for at least one mrna marker using RT PCR than using the CellSearch System (P=0.001) or the AdnaTest (P<0.001). The author s observed a substantial variation in the detection rates of CTCs in blood from breast cancer patients using three different techniques. A higher rate of positive samples was observed using a combined qrt-pcr approach for CK-19 and mammaglobin, which suggests that this is currently the most sensitive technique for detecting CTCs. However, further studies are needed to clarify the prognostic value of this highly sensitive and standardized qrt-pcr approach for CTC detection in peripheral blood of patients with breast cancer. Nakamura et al. (2010) completed a prospective, blinded study and evaluated CTC as a surrogate marker for treatment response and predictor of OS in patients with metastatic breast cancer (n=119; median age 55 years, range 32 to 74). Patients were tested for CTCs at baseline, after 1 cycle of therapy (3 to 4 weeks after baseline), and at 12 weeks follow-up. Patients were included if they were 20 years of age, had progressive, measurable metastatic breast cancer, started a new systemic therapy, and had an Eastern Cooperative Oncology Group (ECOG) performance status core of 0 to 2. All patients underwent CT and/or MRI scans of the chest and abdomen, and a whole body bone scan, in addition to the baseline blood Circulating Tumor Cells Jun 17 15