GPT 3.5 / CX BLADDER SUMMARY, THEN NOVITAS TEXT SECTIONS

CHAT 3.5: INPUT DISCUSSION FROM NOVITAS L39365, AT BOTTOM.

Based on the analysis provided, it appears that the Cxbladder tests, including Cxbladder Detect, Triage, Monitor, and Resolve, have certain limitations and weaknesses in their test design and validation. The foundation of these tests relies on gene expression profiling and the ability to differentiate urothelial carcinoma from other cancerous or non-cancerous conditions. However, there are concerns regarding the ability of the tests to accurately distinguish between different types of malignancies and their potential for false positives.

The analysis points out that the studies validating the Cxbladder tests often exclude patients with certain types of malignancies or inflammatory conditions, which may introduce bias and limit the generalizability of the test results. Furthermore, there is insufficient data on the performance of the Cxbladder tests in the presence of other urinary tract cancers or metastases, which could confound the test results.

The analysis also highlights that the studies funded or performed by Pacific Edge Diagnostics, the company behind Cxbladder, may have potential conflicts of interest. It suggests that the validation studies may not adequately address the limitations and potential confounding factors associated with the tests.

Additionally, the analysis points out that the positive predictive value (PPV) of the Cxbladder tests tends to be low, and there is a high number of false positives, which can lead to patient anxiety and unnecessary follow-up procedures.

Considering these findings, the conclusion drawn in the analysis is that the Cxbladder line of tests, including Cxbladder Detect, Triage, Monitor, and Resolve, is not medically reasonable and necessary for Medicare patients.

Please note that the analysis provided here is based on the information provided in the text you shared and should be evaluated in the context of a comprehensive assessment of the available evidence and medical guidelines.

 

CX BLADDER EVIDENCE  L36395

Summary of Evidence for Specific Lab Tests

Cxbladder (Detect, Triage, Monitor, Resolve)

PubMed and Google Scholar were searched for peer-reviewed, evidence-based literature which provided information regarding analytic and clinical validity and clinical utility for the Cxbladder test. Key words used to search in combination included: Cxbladder, Cxbladder detect, Cxbladder triage, Cxbladder monitor, molecular testing, urine test, bladder cancer, urine biomarker(s), mRNA, uRNA, gene expression profile test, GEP test, 5 gene expression assay, prognostic, and TERT and FGFR3 mutations. Outside of publications from Pacific Edge Diagnostics or studies with funding from that company, only a few peer-reviewed papers have been published addressing the performance of Cxbladder tests.

The Cxbladder line of tests are currently represented by six variations on a five gene expression assay: Cxbladder Detect, Cxbladder Triage, Cxbladder Monitor, Cxbladder Resolve, enhanced Cxbladder Triage, and enhanced Cxbladder Detect. The sequential development of each test variant may be traced through a series of publications, beginning in 2008 with a paper describing the development of Cxbladder’s precursor, the uRNA test, a four gene expression profile (GEP) test.39 Each of the Cxbladder tests rely on a different set of statistical parameters to optimize the function of the five gene expression assay, sometimes synthesizing gene expression data with other inputted data such as patient demographics, cancer history, other clinical history, and single nucleotide variants associated with the FGFR3 and TERT genes. Cxbladder Detect is optimized for sensitivity and specificity as initially described in the seminal 2012 paper.40 Both Cxbladder Triage and Cxbladder Monitor are optimized for sensitivity, Negative Predictive Value (NPV), and test negative rate as initially described in 2015 and 2017 papers, respectively.41,42 Cxbladder Resolve, with a published validation in 2021, is optimized for sensitivity and specificity.43 Most recently, in 2022, the enhanced versions of Cxbladder Triage and Detect were described, with the basic purpose of each parent test unchanged but the parameters of each new test modified by adding data from sequencing six “single nucleotide polymorphisms” associated with two genes: FGFR3 and TERT.44

In 2008, Holyoake and colleagues described a precursor to the Cxbladder tests, uRNA.39 The four gene (CDC2, MDK, IGFBP5, and HOXA13) expression profile test was designed to detect and characterize transitional cell carcinoma (TCC) from patients’ urine. Development of the test involved selection of RNA expression markers that best detected and characterized both early and late stage TCC tumors. The best candidate markers were identified through comparison of tissue from 58 tumors of different stages (Ta-T4) and normal urothelial tissue. Validation of the test utilized urine samples from a cohort of 142 patients that was comprised of 75 patients who were diagnosed with Ta-T4 tumors and 77 “control” patients. The overall specificity of this test was 85% with a range of sensitivities depending on tumor stage (from 48% for Ta tumors to 100% for tumors with a stage greater than T1).

In 2012, O’Sullivan and colleagues developed and validated the first Cxbladder test (Cxbladder Detect), using a foundation of the uRNA four GEP test and adding an additional gene (CXCR2) to this profile.40 The 2012 publication also compared the new Cxbladder test to its precursor test (uRNA-D), urine cytology, and other urine tests on the market (NMP22 ELISA and BladderChek). The patient cohort was comprised of 485 patients presenting with gross hematuria. Cxbladder demonstrated an 81.8% sensitivity at a fixed specificity of 85%; all other tests in the comparison fell below a sensitivity of 65%, although the specificity of all other tests was higher than Cxbladder, with the highest specificity at 96.4% for the BladderChek test.

In 2015, Breen and colleagues further evaluated the Cxbladder Detect test in a comparative study with other tests used to detect urothelial carcinoma in urine.45 The other tests evaluated included cytology, UroVysion FISH, and NMP22. The study utilized five cohorts of patients, only one of which evaluated all four tests for the entire cohort. Data from the five cohorts were evaluated and integrated, with several different imputation analyses utilized to fill in for missing test values and create a “new, imputed, comprehensive dataset.” From this data, the authors found that before imputation, Cxbladder Detect had superior sensitivity (79.5%) as compared to the other three tests (the second highest sensitivity being 45.5%) but inferior specificity (82.2%), with the second lowest specificity being 87.3%. Utilizing several different imputation methodologies, similar findings for comparative sensitivities and specificities were seen, leading to the conclusion that the imputed data sets were valid, with the best imputation methodology being the 3NN model. Finally, with the new imputed data set, the authors re-assessed the comparisons between tests and found that Cxbladder Detect “outperformed” the other tests in this study.

In 2015, Kavalieris and colleagues developed another version of the Cxbladder test (later to be called Cxbladder Triage), this time evaluating the impact of adding clinical data (age, gender, frequency of macrohematuria, and smoking history) to the testing algorithm.41 Genetic input into the algorithm was termed the G INDEX while clinical data was termed the P INDEX. The study utilized 517 patients with macrohematuria from the 2012 Cxbladder study population, an additional 178 patients with macrohematuria from two separate cohorts, and 45 patients from a small cohort of patients with microhematuria.38 Combining the G and P indices provided a better bias-corrected receiver operating characteristic curve (AUC) (0.86) than either of the indices alone (0.83 and 0.61 respectively). When set at a test-negative rate of 0.4, the G + P INDEX performed with a sensitivity of 95% and NPV of 98%, improving on the G INDEX sensitivity and NPV of 86% and 96% respectively. The authors envisioned the G + P INDEX being used to triage outpatients with a low probability of having urothelial carcinoma, reducing the need for diagnostic procedures.

In 2017, Kavalieris and colleagues developed another version of the Cxbladder test (Cxbladder Monitor) utilizing a cohort of 763 patients under surveillance for recurrence of bladder urothelial carcinoma.42 In addition to the data from the five gene expression profile, Cxbladder Monitor also used clinical data in its algorithm which included previous tumor status (primary tumor or recurrent tumor) and the number of years elapsed since the previous tumor. The paper analyzed several subgroups including different stages of tumor and patients who had received adjuvant bacillus Calmette-Guerin (BCG) treatment. With a test negativity rate of 0.34, Cxbladder Monitor demonstrated a sensitivity of 93% and NPV of 97%.

Also from 2017, Lotan and colleagues utilized the same patient cohort found in the Kavalieris (2017) study to perform a comparative analysis between Cxbladder Monitor and other noninvasive urine tests that were used to rule out recurrent urothelial carcinoma.42,46 The authors found that Cxbladder “outperformed” all comparative tests (which included cytology, NMP22 ELISA, NMP22 BladderChek, and UroVysion FISH), with higher sensitivity (91% versus sensitivities ranging from 11% to 33%) and higher NPV (96% versus NPVs ranging from 86% to 92%).

In 2017, Darling and colleagues performed a clinical utility study for Cxbladder Triage and Detect.47 The study used previously obtained clinical data and Cxbladder test results to create clinical scenarios for twelve urologists. These scenarios centered on patients presenting with hematuria and evaluated how the urologists would hypothetically work-up these patients in the context of Cxbladder results. The study found that when the urologists had access to Cxbladder results, they would hypothetically change their clinical decisions in caring for these patients, ultimately leading to fewer invasive diagnostic procedures.

In 2018, Lough and colleagues performed a clinical utility study for Cxbladder Monitor.48 The study used previously obtained clinical data and Cxbladder test results to create clinical scenarios for eighteen physicians. These scenarios centered on patients with a history of urothelial carcinoma and evaluated how the physicians would hypothetically manage these patients in the context of Cxbladder results. The study found that when the physicians had access to Cxbladder results, they would hypothetically change their clinical decisions in caring for these patients, leading ultimately to fewer tests and procedures for patients classified as low risk by Cxbladder and an increased number of tests and procedures for patients classified as higher risk.

In 2019, Konety and colleagues performed a retrospective analysis of pooled data (from four patient cohorts) in the context of Cxbladder Triage, Detect, and Monitor.49 A total of 436 samples were evaluated from patients with hematuria and 416 samples from patients with potential recurrence of urothelial carcinoma. These Cxbladder results were then compared with cytology results for the same samples. The authors found that overall, Cxbladder demonstrated a better NPV than cytology (97.4% versus 92.6%) and missed less tumors (false negatives) than cytology (eight missed versus 59 missed).

In 2020, Koya and colleagues performed a retrospective audit of a new surveillance protocol that incorporated Cxbladder Monitor for patients with a history of urothelial carcinoma.50 The patients involved were divided into two cohorts: low risk (n = 161) and high risk (n = 47), noting that these numbers represent only patients who completed the study with both Cxbladder testing and follow up cystoscopy. There were 309 patients who were initially enrolled in the study but only 208 that completed the study. In the low-risk cohort, patients who received a negative result for the Cxbladder test were permitted to wait longer (12 months as opposed to within two to three months of a positive result) before receiving a follow-up cystoscopy. In the high-risk cohort, patients were managed the same regardless of the Cxbladder result, although the data was used to speculate on potential changes to the protocols for this type of patient. Over the course of the study and in the 35 months of the follow-up period, no cases positive for urothelial carcinoma were missed by the first Cxbladder test (although there was at least one false negative result in a second, follow-up round of Cxbladder testing) and no patients developed newly invasive or metastatic urothelial carcinoma. For the low-risk cohort, confirmed recurrence occurred in three of the patients who initially tested negative in the Cxbladder test; only two of those three patients had a follow-up, second Cxbladder test, with one true positive and one false negative (as demonstrated in the supplementary figures). There was confirmed recurrence in three low risk patients who tested positive with Cxbladder. For high risk patients, four patients demonstrated a recurrence of urothelial carcinoma and all four of these patients tested positive with Cxbladder.

In 2023, Li and colleagues evaluated Cxbladder Monitor through a prospective study of 92 patients diagnosed with non-muscle invasive bladder cancer (NMIBC) from two different clinical sites that were ready for follow up regarding their diagnosis (primary or recurrent), a previous procedural visit, and/or other therapy (e.g., BCG instillation).51 The study sought to triage scheduling these patients’ for follow-up cystoscopy through use of at home Cxbladder Monitor testing, delaying the follow-up appointment if patients received a lower risk score (<3.5) on the Monitor test. Patients with either gross hematuria or active UTI were excluded from the study. Moreover, of the 92 patients, a total of 16 were lost to follow-up, although data was still included in summary tables for these 16 patients. Of the 24 patients followed-up earlier due to a higher risk score (>3.5), nine were found to have tumors on cystoscopy. Of the 52 patients with delayed cystoscopy due to a lower risk score, none were found to have tumor. Note that of the 66 patients with a lower risk score, 14 were not evaluated via the delayed cystoscopy for the following reasons: did not show up for cystoscopy, chose another round of Cxbladder Monitor testing instead of cystoscopy, stopped surveillance for reasons not given, or died of “unrelated” but undescribed causes. The paper also noted that the patients who opted out of the follow-up cystoscopy in favor of a second Cxbladder Monitor test were only found at one of the two sites. The authors concluded that using at-home Cxbladder Monitor testing to triage patients and allow delayed cystoscopy for patients with a lower risk score was an effective new protocol.

In 2021, Raman and colleagues developed a fourth version of the Cxbladder test, Cxbladder Resolve, utilizing three different patient cohorts (total of 863 patients) in the internal validation and one separate cohort (548 patients) in the external validation.43 In the external validation, testing was also performed with other versions of the Cxbladder test: Cxbladder Triage and Detect.

Cxbladder Resolve was designed to identify patients with a high probability of high-impact tumors (HIT), namely high grade urothelial carcinomas, by stratifying patients into one of three categories: high priority for HIT evaluation, work-up for HIT based on physician-directed protocol (PDP), or manage by observation. In the internal validation, Cxbladder Resolve was found to have a bootstrap-adjusted estimated sensitivity of 92.4% and specificity of 93.8% for HIT; note that the overall sensitivity and specificity for all tumors during the internal validation was 91.2% and 61.0% respectively. In the external validation, Cxbladder Resolve correctly identified all HIT diagnoses and missed three low grade tumors, with a cumulative sensitivity of 90.0% and specificity of 96.3%. The authors also found that using a reflexive test algorithm with Cxbladder Triage, Detect, and Resolve together would correctly identify 87.6% of patients who did not need further work-up (NPV of 99.4%).

In 2022, Lotan and colleagues published a study describing two newer versions of the Cxbladder tests (enhanced Cxbladder Triage [CxbT+] and Detect [CxbD+]).46 The enhancement (digital droplet PCR testing of urine specimens) was used to identify the presence or absence of six single nucleotide polymorphisms (SNPs) associated with the genes FGFR3 and TERT. These SNPs, mostly somatic (acquired) genetic variants, can be found in urothelial carcinomas, as described in the literary references provided in Lotan and colleagues’ paper. Lotan and colleagues performed an internal validation of these new biomarkers, testing urine from two cohorts: 344 patients from the United States and 460 patients from Singapore. The six SNPs were evaluated both as stand-alone tests and as enhancement to original Cxbladder Triage and Detect tests. The authors concluded that the addition of the six SNPs to the Cxbladder tests improved test performance, particularly specificity.

Two publications from Davidson and colleagues in 2019 and 2020 evaluated the performance of the Cxbladder Triage test when integrated into hematuria work-up protocols.52,53 Notably, these studies were not performed or funded by Pacific Edge Diagnostics. The 2019 paper prospectively evaluated the new protocol without enacting it within the clinical setting while the 2020 paper described the outcome of a fully enacted protocol. In the 2019 study, which included 478 patients with hematuria referred to the urology practice and 73 patients with hematuria who had not been referred, the Cxbladder test correctly triaged 42 of 44 patients with urothelial malignancy; the two false negatives were either confirmed or suspected (no histology obtained) low grade lesions. From their cohort, the authors found that Cxbladder Triage had a sensitivity of 95% and NPV of 98%. The authors concluded “the risk of missing a significant cancer from the adoption of the theoretical pathway appears very low and clinically acceptable,” while later stating that larger studies were still needed to “prove the true clinical value of inclusion of these biomarkers in investigative pathways.” In the 2020 study, Davidson and colleagues retrospectively evaluated the clinical courses of 884 patients with hematuria who were worked-up with the new protocol and subsequently followed for a median of 21 months. The protocol identified 46 histologically confirmed urothelial carcinomas. Cxbladder Triage results included five false negatives, four of which were detected upon imaging and one of which was discovered in a three month follow up. Cxbladder Triage results also demonstrated low specificity with 39% of results being false positives. Overall, the authors found that the protocol that included Cxbladder Triage had a sensitivity of 98.1% and NPV of 99.9%. The authors concluded that their findings “add to the increasing evidence that biomarkers have a place in the assessment of hematuria, but that the results of these assays need to be supported by imaging of the bladder.”

In terms of systemic reviews and meta-analyses, two publications in 2015 were identified, both from Chou and colleagues, in contract with the Agency for Healthcare Research and Quality, AHRQ, U.S. Department of Health and Human Services.54,55 The publications discussed several urinary biomarker tests including Cxbladder Detect. However, both publications only discussed a single Cxbladder study performed by O’Sullivan and colleagues in 2012.40 In the shorter publication by Chou and colleagues, a systematic review and meta-analysis in the Annals of Internal Medicine, the sensitivity and specificity of Cxbladder was given a low grade for strength of evidence (as determined by study quality, precision, consistency and directness). The process of evidence assessment was covered in greater detail in the 923 page document from Chou and colleagues entitled “Emerging Approaches to Diagnosis and Treatment of Non-Muscle-Invasive Bladder Cancer;” however, the key points concerning Cxbladder were covered in the shorter publication and mostly just reiterated in the longer document.

Another systemic review and meta-analysis was published more recently in 2022 by Laukhtina and colleagues.56 The study reviewed five different urinary biomarker tests (UBT) used to detect recurrent urothelial carcinoma, including Cxbladder Monitor. The authors assessed statistical values associated with each test, such as sensitivity, specificity, positive predictive value (PPV), NPV, and accuracy. Additionally, the authors assessed the risk of bias using the Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS-2), using cystoscopy and histology results for reference. The study also performed network meta-analysis on the tests as compared with cytology. Two Cxbladder Monitor studies were analyzed: Koya (2020) and Lotan (2017).46,50 At the end of the paper, the authors concluded the performances of the five tests “support[ed] their potential value in preventing unnecessary cystoscopies.” The authors also assessed other diagnostic tests from four of the five test companies, including Cxbladder Triage and Detect (O’Sullivan [2012] and Davidson [2019]) and concluded “there are not enough data to support their use in the initial diagnosis setting.”40,52

CX CXBLADDER: ANALYSIS & CONCLUSIONS

Cxbladder (Detect, Triage, Monitor, Resolve)

The fundamental methodology of Cxbladder tests is founded on a 2008 paper from Holyoake and colleagues describing the creation of an RNA expression assay that could predict the likelihood of urothelial carcinoma from urine.39 Several gene expression profiles were examined between urothelial carcinoma tissue and normal urothelial tissue, the latter of which was collected as non-malignant tissue from patients with renal cell carcinoma who had undergone a radical nephrectomy. The gene expression profiles that demonstrated the most promise in differentiating between cancer and non-cancer were gathered into a four gene expression panel and then optimized to discriminate between urine from patients with any grade/stage of urothelial cancer and patients without urothelial cancer. Gene expression tests that are used to predict the presence or absence of cancer, however, must take into consideration many potential complicating and confounding factors. The absence of a rigorous approach to addressing complicating/confounding factors undermined the clinical validity of Cxbladder tests, as will be detailed below.

As evidenced in the publications reviewed in the Summary of Evidence, the key weakness of the Cxbladder tests is found within their test design. Cxbladder tests are founded on the concept that differences in gene expression between urothelial cancer and non-urothelial cancer (including non-neoplastic tissue) can be measured in urine to determine if urothelial cancer is present or not present. This means that a well-designed test will be able to not only discriminate between cancer and normal tissue, but also between different types of malignancy.

For the precursor test uRNA, Holyoake and colleagues started with a custom-printed array from MWG Biotech that allowed gene expression profiling of 26,600 genes.39 This array was used to analyze normal tissue (18 specimens) and urothelial carcinoma tissue (28 specimens from Ta tumors and 30 specimens from T1-T4 tumors). The preliminary data was then analyzed to select the most promising genes for creation of a GEP test. This subset of promising genes was further pruned by testing urine from patients with transitional cell carcinoma (TCC) (urothelial carcinoma) (n=75), patients with other “urological cancer” (n=33), and patients without cancer, including patients with infection (n=20) and “other benign urinary tract disease” (n=24). Additionally, the paper mentions testing blood to get gene expression levels for blood and inflammatory cells, but the results of this subset of tests were not provided in this paper. It must be noted here that the characteristics of the non-TCC cancers were also not disclosed in the paper.

After this additional testing, Holyoake and colleagues settled on four gene expression test (uRNA-D) that utilized the genes MDK, CDC2 (now officially known as CDK1), IGFBP5, and HOXA13.39 Unfortunately, the false positives and false negatives received little attention from the paper, including false positives for patients with other non-TCC cancers (n=3). The authors concluded that their results “will need to be further validated in a prospective setting to more accurately determine test characteristics, particularly in patients presenting with hematuria and other urological conditions.”

Standing alone, the 2008 paper from Holyoake and colleagues lacks the scientific rigor to establish the uRNA-D test as able to accurately distinguish between urothelial carcinoma and other cancers or other non-cancer urological conditions.39 One very notable gap included a lack of details or definition for non-urothelial cancers, of which many would feed into the urinary system, including prostate cancers, renal cancers, and metastatic or locally invasive cancers from other organs. It would be expected that a well-designed test would assess not only the full spectrum of potential cancers, but that the test design would include a much higher count of specimens (beyond the 33 undefined cancers found in this study). In the same sense, the absence of details regarding non-malignant specimens, which included only 20 “urinary tract infections,” was a major and notable gap in this test’s development. There were many other issues identified with this paper including a strong population bias towards male patients, but altogether, this validation of uRNA-D was insufficient to support that the test performed accurately as a tool for distinguishing between urine from patients with and without urothelial carcinoma.

It is critical to understand the limitations of the 2008 publication from Holyoake and colleagues because the test uRNA-D was used to create the Cxbladder line of tests, with the main difference between uRNA-D and Cxbladder being the addition of a single gene, CXCR2, to the gene expression profile of the Cxbladder assay.39,40 It is noted that other versions of Cxbladder use non-genetic data in an overarching algorithm to produce results, but the focus of this discussion will be upon the gene expression profile technology of Cxbladder tests.

In 2012, the first paper describing a Cxbladder test was published by O’Sullivan and colleagues.40 This paper acted as both a test validation and a comparison of the new Cxbladder test with other urine tests on the market. While the statistical results of Cxbladder seem promising, we must return to the foundation of the test, namely its ability to distinguish between urothelial carcinoma and other cancerous or non-cancerous conditions (or patients without disease). In this paper, other malignancies (n=7) were assessed only when they were found in patients with urothelial carcinoma. Moreover, the types of other malignancy were not disclosed in this paper. There are also 255 “nonmalignant disease” specimens, which included representations of “benign prostatic hyperplasia/prostatitis”, “cystitis/infection or inflammation of urinary tract”, calculi, and “hematuria secondary to warfarin,” and 164 specimens from patients with “no specific diagnosis.” This first paper from 2012 also does not sufficiently address Cxbladder’s ability to distinguish between urothelial carcinoma and other malignancies, which is of particular relevance when a majority of the patient population were male (78%) with a median patient age of 64 years and thus, with higher risk of prostate carcinoma. The paucity of clinical data also created gaps in the data integrity, failing to answer questions such as how many of the urothelial carcinoma specimens had coincident inflammation and what other medical conditions (and medications) were present in this patient population? Additionally, the paper does not spend significant time discussing the potential reasons for false positives and false negatives. These issues are compounded by a short follow-up period (only 12-months) with participating patients.

In the most recent paper published for Pacific Edge Diagnostics by Lotan and colleagues in 2022, Cxbladder Triage and Detect were “enhanced” by addition of a different test methodology, digital droplet PCR, adding a different approach to detecting urothelial carcinoma: identification genetic variants associated with urothelial carcinoma.44 This approach was based on the premise that the six variants (called single nucleotide polymorphisms or SNPs in the paper) are either acquired as mutations in the carcinogenesis of urothelial carcinoma or already present as an inherited genetic variant in the patient’s germline DNA, representing a higher risk of urothelial carcinoma. However, it is known that these SNPs can also show up in the context of other malignancies (such as papillary renal cell carcinoma), which is not addressed by Lotan and colleagues.183,184 Moreover, as mentioned in the paper’s discussion, the presence of these SNPs in urine may not coincide with clinically detectable (e.g., cystoscopically visible) carcinoma. This could lead to further confusion with false positives, especially when the PPV of Cxbladder tests tends to be very low. If numerous false positive results in Cxbladder are accepted as an inherent trait of the test, providers may not be as vigilant in closely following patients with a positive Cxbladder result after a negative cystoscopy. In addition, providers may not search for other malignancies (e.g., papillary renal cell carcinoma) as a potential cause for the “false positive” Cxbladder result. Another weakness of the 2022 study was seen in the differences between cohorts. Notably, the six SNPs alone were less sensitive for urothelial carcinoma in the Singapore cohort (66%) than in the United States cohort (83%). This could indicate differences in the genetic etiology of urothelial carcinoma in different populations, meaning that the six SNPs may not be as representative in populations not evaluated in this study. Furthermore, while this study claimed to evaluate multiple ethnicities, the paper does not disclose which ethnicities were evaluated and the numbers of patients from each ethnicity.

Each new Cxbladder test builds on their predecessors, often utilizing the same specimens from prior studies in their test validations and performance characterizations. Moreover, the insufficient assessment of potential confounding factors is perpetuated through these studies. For instance, if we look at assessment of non-urothelial cancer through all major published uDNA and Cxbladder studies, we see the following:

• Holyoake 2008: 33 undefined cancers were noted39

• O’Sullivan 2012: Seven other malignancies (undefined) were noted, all in patients with urothelial carcinoma40

• Kavalieris 2015: Non-urothelial neoplasms were not discussed (study population included 517 patients from the O’Sullivan 2012 study)40,41

• Breen 2015: Non-urothelial neoplasms were not discussed (study population included patients from the O’Sullivan 2012 study)40,45

• Kavalieris 2017: Non-urothelial neoplasms were not discussed (same patient population as Lotan 2017)42,46

• Lotan 2017: Non-urothelial neoplasms were not discussed (same patient population as Kavalieris 2017) 42,46

• Konety 2019: In some subpopulations, patients with history of prostate or renal cell carcinoma were excluded from the study; otherwise, non-urothelial neoplasms were not discussed (study population included patients from O’Sullivan 2012 and Kavalieris/Lotan 2017) 40,42,46,49

• Davidson 2019: Other non-bladder malignancies and neoplasms were identified (but not subclassified) in a study evaluating hematuria; notably, Cxbladder-Triage was positive in most of these other malignancies (seven of nine total) and neoplasms (two of three total)52

• Koya 2020: Non-urothelial neoplasms were not discussed50

• Davidson 2020: Other non-bladder malignancies and neoplasms were identified in the study but data was not presented to allow association of these other malignancies and neoplasms with positive or negative results from Cxbladder.53

• Raman 2021: In some subpopulations, patients with history of prostate or renal cell carcinoma were excluded from the study; otherwise, non-urothelial neoplasms were not discussed (study population included patients from O’Sullivan 2012 and Konety 2021)40,43,49

• Lotan 2022: In some subpopulations, patients with history of prostate or renal cell carcinoma were excluded from the study; otherwise, non-urothelial neoplasms were not discussed44

• Li 2023: Some patients (24 of 92 patients) noted to have “other cancers;” except for one mention (a patient with breast cancer who missed their nine month follow-up due to conflict with breast cancer treatment), other types of cancers are not described or significantly discussed51

There are numerous potential malignancies that can contribute to urine genetic composition (e.g., renal cell cancer, bladder cancer, prostate cancer). However, by using only 40 unspecified neoplasm specimens, 33 of which were tested only for uDNA (not Cxbladder), the validation from Pacific Edge Diagnostics underrepresents potentially confounding variables. This underrepresentation is further substantiated through data found in studies not performed or funded by Pacific Edge Diagnostics. In an independent study from Davidson and colleagues in 2019 performed on patients with hematuria, seven of nine malignant prostate or kidney lesions were discovered in patients with a positive Cxbladder-Triage result.52 False positive Cxbladder-Triage results were also seen in a majority of patients without bladder cancer but instead diagnosed with radiation cystitis, vascular prostate, bladder stones, anticoagulation related bleeding, post-TURP bleeding, and urethral stricture. No cause for hematuria was found in 225 patients, with 137 of them having a positive Cxbladder-Triage result. This 2019 study as well as others indicate that Cxbladder is less sensitive for detecting smaller, low grade malignancies making it unlikely the false positives could represent urothelial malignancies below the limit of detection by cystoscopy and other conventional evaluations.52

The exclusion criteria further weakened the development and validation of Cxbladder tests. Consistent with the aforementioned confounding variables of other urinary tract cancers and metastases, the Cxbladder studies generally excluded patients with a history of prostate or renal cancer; however, these exclusions were not always seen in studies funded and/or published by Pacific Edge Diagnostics. The validation studies for Cxbladder also typically excluded inflammatory disorders such as pyelonephritis and active urinary tract infections and also excluded known causes for hematuria like bladder or renal calculi or recent manipulation of the genitourinary tract (e.g., cystoscopy).40-43, 46 In the first published validation study for Cxbladder, the authors stated that the additional 5th RNA marker, CXCR2, “was predicted to reduce the risk of false-positive results in patients with acutely or chronically inflamed urothelium.”40 However, in this same study, the authors went on to exclude patients with “documented urinary tract infection.” Fortunately, publications from other sources such as Davidson and colleagues in 2019 provide insight into how Cxbladder tests (namely Cxbladder Triage) perform under these benign conditions.52 Davidson and colleagues found that false positives were seen in a majority of patients where the underlying etiology of hematuria was radiation cystitis, vascular prostate, bladder stones, anticoagulation related bleeding, post-TURP bleeding, or urethral stricture. False positives were also seen in 10 of 23 (43%) patients with urinary tract infection and 8 of 10 (80%) patients with “other” inflammatory etiologies. Altogether, in the inflammatory category of the study, over half (59%) of patients with an inflammatory etiology of their hematuria received a false positive result from the Cxbladder Triage (CxbT) test. In a subsequent study by Davidson and colleagues in 2020, “approximately 10% of patients (85 of 884) required a repeat CxbT assay because quality control failures, mainly caused by interference of inflammatory products or a large number of white blood cells.”53 The couple of systemic reviews and meta-analyses that included Cxbladder tests were mixed in their assessment of this line of tests. Chou and colleagues in 2015 only reviewed one of the Cxbladder papers (O’Sullivan 2012) and came to the conclusion that the strength of evidence for the study was graded low.40,54,55 In 2022, Laukhtina and colleagues performed a more involved assessment of Cxbladder tests, particularly Cxbladder Monitor, coming to the conclusion that it had “potential value in preventing unnecessary cystoscopies.”56 The authors also determined that there was “not enough data to support” using Cxbladder Triage and Detect in the “initial diagnosis setting.” Laukhtina and colleagues did acknowledge that their study had several potential limitations which included the “absence of data on blinding to pathologist and urologists” and the inability to “perform subgroup analyses for HG [High Grade urothelial carcinoma] recurrence detection only.” However, it should be noted that for both the 2015 and 2022 systemic reviews and meta-analyses, the evaluation of the actual clinical features of each Cxbladder study was relatively superficial, focusing more on the statistical values and less on the quality of the studies and the designs underlying those values.54-56

In conclusion, the Cxbladder line of tests all suffer from the foundational problem of insufficient validation of their test in potentially confounding clinical circumstances including non-urothelial carcinoma malignancies and inflammatory conditions of the urinary tract. Cxbladder also demonstrates several population biases, including early papers with a strong bias towards male patients of European ancestry. The majority of Cxbladder papers avoid disclosing the PPV and number of false positives of their tests. Cxbladder tests generally have low PPVs (down to 15-16% as seen in Konety, et al 2019) and high numbers of false positives (also in Konety, et al 2019, there were 464 false positive results as compared to 86 true positive results).49 These values are significant in that false test results, particularly false positives, can lead to patient anxiety and distress among other procedural issues related to follow up for an inaccurate result. Most of the primary literature regarding Cxbladder test development and performance is funded, if not directly written by, the test’s parent company, Pacific Edge Diagnostics. This conflict of interest must be taken into account when reviewing these papers. Finally, and most importantly, due to the insufficient representation of confounding factors in the validation populations, the Cxbladder tests have not been adequately vetted in the context of the Medicare population. Given all of these findings, the Cxbladder line of tests are considered not medically reasonable and necessary for Medicare patients.