Development of a multigenomic liquid biopsy (PROSTest) for prostate cancer in whole blood.

INTRODUCTION: We describe the development of a molecular assay from publicly available tumor tissue mRNA databases using machine learning and present preliminary evidence of functionality as a diagnostic and monitoring tool for prostate cancer (PCa) in whole blood. MATERIALS AND METHODS: We assessed 1055 PCas (public microarray data sets) to identify putative mRNA biomarkers. Specificity was confirmed against 32 different solid and hematological cancers from The Cancer Genome Atlas (n = 10,990). This defined a 27-gene panel which was validated by qPCR in 50 histologically confirmed PCa surgical specimens and matched blood. An ensemble classifier (Random Forest, Support Vector Machines, XGBoost) was trained in age-matched PCas (n = 294), and in 72 controls and 64 BPH. Classifier performance was validated in two independent sets (n = 263 PCas; n = 99 controls). We assessed the panel as a postoperative disease monitor in a radical prostatectomy cohort (RPC: n = 47). RESULTS: A PCa-specific 27-gene panel was identified. Matched blood and tumor gene expression levels were concordant (r = 0.72, p < 0.0001). The ensemble classifier ("PROSTest") was scaled 0%-100% and the industry-standard operating point of ≥50% used to define a PCa. Using this, the PROSTest exhibited an 85% sensitivity and 95% specificity for PCa versus controls. In two independent sets, the metrics were 92%-95% sensitivity and 100% specificity. In the RPCs (n = 47), PROSTest scores decreased from 72% ± 7% to 33% ± 16% (p < 0.0001, Mann-Whitney test). PROSTest was 26% ± 8% in 37 with normal postoperative PSA levels (<0.1 ng/mL). In 10 with elevated postoperative PSA, PROSTest was 60% ± 4%. CONCLUSION: A 27-gene whole blood signature for PCa is concordant with tissue mRNA levels. Measuring blood expression provides a minimally invasive genomic tool that may facilitate prostate cancer management.

Update of the diagnostic and therapeutic guidelines for gastro-entero-pancreatic neuroendocrine neoplasms (recommended by the Polish Network of Neuroendocrine Tumours) [Aktualizacja zalecen ogólnych dotyczacych postepowania diagnostyczno-terapeutycznego w nowotworach neuroendokrynnych ukladu pokarmowego (rekomendowane przez Polska Siec Guzów Neuroendokrynnych)].

Continuous progress in the diagnostics and treatment of neuroendocrine neoplasms (NENs), the emerging results of new clinical trials, and the new guidelines issued by medical societies have prompted experts from the Polish Network of Neuroendocrine Tumours to update the 2017 recommendations regarding the management of neuroendocrine neoplasms. This article presents the general recommendations for the management of NENs, resulting from the findings of the experts participating in the Fourth Round Table Conference, entitled "Polish Guidelines for the Diagnostics and Treatment of Neuroendocrine Neoplasms of the gastrointestinal tract, Zelechów, June 2021". Drawing from the extensive experience of centres treating these cancers, we hope that we have managed to formulate the optimal method of treating patients with NENs, applying the latest reports and achievements in the field of medicine, which can be effectively implemented in our country. The respective parts of this work present the approach to the management of: NENs of the stomach and duodenum (including gastrinoma), pancreas, small intestine, and appendix, as well as large intestine.

Pancreatic neuroendocrine neoplasms - update of the diagnostic and therapeutic guidelines (recommended by the Polish Network of Neuroendocrine Tumours) [Nowotwory neuroendokrynne trzustki - uaktualnione zasady diagnostyki i leczenia (rekomendowane przez Polska Siec Guzów Neuroendokrynych)].

In this paper, we present the current guidelines for the diagnostics and management of pancreatic neuroendocrine neoplasms (PanNENs) developed by Polish experts providing care for these patients in everyday clinical practice. In oncological diagnostics, in addition to biochemical tests, molecular identification with the use of NETest liquid biopsy and circulating microRNAs is gaining importance. Both anatomical and functional examinations (including new radiopharmaceuticals) are used in imaging diagnostics. Histopathological diagnosis along with immunohistochemical examination still constitute the basis for therapeutic decisions. Whenever possible, surgical procedure is the treatment of choice. Pharmacological management including biotherapy, radioisotope therapy, targeted molecular therapy and chemotherapy are important methods of systemic therapy. Treatment of PanNENs requires a multidisciplinary team of specialists in the field of neuroendocrine neoplasms.

Gastroduodenal neuroendocrine neoplasms including gastrinoma - update of the diagnostic and therapeutic guidelines (recommended by the Polish Network of Neuroendocrine Tumours) [Nowotwory neuroendokrynne zoladka i dwunastnicy z uwzglednieniem gastrinoma - uaktualnione zasady postepowania (rekomendowane przez Polska Siec Guzów Neuroendokrynnych)].

After another meeting of experts of the Polish Network of Neuroendocrine Tumours, updated recommendations for the management of patients with gastric and duodenal neuroendocrine neoplasms, including gastrinoma, have been issued. As before, the epidemiology, pathogenesis and clinical symptoms of these neoplasms have been discussed, as well as the principles of diagnostic procedures, including biochemical and histopathological diagnostics and tumour localisation, highlighting the changes introduced in the recommendations. Updated principles of therapeutic management have also been presented, including endoscopic and surgical treatment, and the options of pharmacological and radioisotope treatment. The importance of monitoring patients with gastric and duodenal NENs, including gastrinoma, has also been emphasised.

Colorectal neuroendocrine neoplasms - update of the diagnostic and therapeutic guidelines (recommended by the Polish Network of Neuroendocrine Tumours) [Nowotwory neuroendokrynne jelita grubego - uaktualnione zasady diagnostyki i leczenia (rekomendowane przez Polska Siec Guzów Neuroendokrynych)].

Colorectal neuroendocrine neoplasm (CRNEN), especially rectal tumours, are diagnosed with increased frequency due to the widespread use of colonoscopy, including screening examinations. It is important to constantly update and promote the principles of optimal diagnostics and treatment of these neoplasms. Based on the latest literature and arrangements made at the working meeting of the Polish Network of Neuroendocrine Tumours (June 2021), this paper includes updated and supplemented data and guidelines for the management of CRNEN originally published in Endokrynologia Polska 2017; 68: 250-260.

Neuroendocrine neoplasms of the small intestine and the appendix - update of the diagnostic and therapeutic guidelines (recommended by the Polish Network of Neuroendocrine Tumours) [Nowotwory neuroendokrynne jelita cienkiego i wyrostka robaczkowego - uaktualnione zasady diagnostyki i leczenia (rekomendowane przez Polska Siec Guzów Neuroendokrynnych)].

Updated Polish recommendations for the management of patients with neuroendocrine neoplasms (NENs) of the small intestine (SINENs) and of the appendix (ANENs) are presented here. The small intestine, and especially the ileum, is one of the most common locations for these neoplasms. Most of them are well-differentiated and slow-growing tumours; uncommonly - neuroendocrine carcinomas. Their symptoms may be untypical and their diagnosis may be delayed or accidental. Najczesciej pierwsza manifestacja ANEN jest jego ostre zapalenie. Typical symptoms of carcinoid syndrome occur in approximately 20-30% of SINENs patients with distant metastases. In laboratory diagnostics the assessment of 5-hydroxyindoleacetic acid concentration is helpful in the diagnosis of carcinoid syndrome. The most commonly used imaging methods are ultrasound examination, computed tomography, magnetic resonance imaging, colonoscopy and somatostatin receptor imaging. Histopathological examination is crucial for the proper diagnosis and treatment of patients with SINENs and ANENs. The treatment of choice is a surgical procedure, either radical or palliative. Long-acting somatostatin analogues (SSAs) are essential in the medical treatment of functional and non-functional SINENs. In patients with SINENs, at the stage dissemination with progression during SSAs treatment, with high expression of somatostatin receptors, radioisotope therapy should be considered first followed by targeted therapies - everolimus. After the exhaustion of the above available therapies, chemotherapy may be considered in selected cases. Recommendations for patient monitoring are also presented.

Molecular Genomic Assessment Using a Blood-based mRNA Signature (NETest) is Cost-effective and Predicts Neuroendocrine Tumor Recurrence With 94% Accuracy.

INTRODUCTION: Identification of residual disease after neuroendocrine tumor (NET) resection is critical for management. Post-surgery imaging is insensitive, expensive, and current biomarkers ineffective. We evaluated whether the NETest, a multigene liquid biopsy blood biomarker, correlated with surgical resection and could predict recurrence. METHODS: Multicenter evaluation of NET resections over 24 months (n = 103): 47 pancreas, 26 small bowel, 26 lung, 2 appendix, 1 duodenum, 1 stomach. Surgery: R0 (83), R1/R2 (20). One millilitre of blood was collected at D0 and posroperative day (POD) 30. Transcript quantification by polymerase chain reaction (normal: ≤20), CgA by NEOLISA (normal ≤108 ng/mL). Standard-of-care (SoC) follow-up costs were calculated and compared to POD30 NETest-stratification approach. Analyses: Wilcoxon-paired test, Chi-square test. D BIOMARKERS: NETest: 103 of 103 (100%)-positive, whereas 23 of 103 (22%) were CgA-positive (Chi-square = 78, P < 0.0001).In the R0 group, the NETest decreased 59 ± 28 to 26 ± 23 (P < 0.0001); 36% (30/83) remained elevated. No significant decrease was evident for CgA. In the R1/R2 group the NETest decreased but 100% remained elevated. CgA levels did not decrease.An elevated POD30 NETest was present in R0 and 25 (83%) developed radiological recurrences. Normal score R0 s (n = 53) did not develop recurrence (Chi-square = 56, P < 0.0001). Recurrence prediction was 94% accurate with the NETest. COST EVALUATION: Using the NETest to stratify postoperative imaging resulted in a cost-savings of 42%. CONCLUSION: NETest diagnosis is more accurate than CgA (100% vs 22%). Surgery significantly decreased NETest. An elevated POD30 NETest predicted recurrence with 94% accuracy and post-surgical POD30 NETest follow-up stratification decreased costs by 42%. CgA had no surgical utility. Further studies would define the accuracy and cost-effectiveness of the NETest in the detection of postoperative recurrent disease.

Early Identification of Residual Disease After Neuroendocrine Tumor Resection Using a Liquid Biopsy Multigenomic mRNA Signature (NETest).

INTRODUCTION: Surgery is the only cure for neuroendocrine tumors (NETs), with R0 resection being critical for successful tumor removal. Early detection of residual disease is key for optimal management, but both imaging and current biomarkers are ineffective post-surgery. NETest, a multigene blood biomarker, identifies NETs with >90% accuracy. We hypothesized that surgery would decrease NETest levels and that elevated scores post-surgery would predict recurrence. METHODS: This was a multicenter evaluation of surgically treated primary NETs (n = 153). Blood sampling was performed at day 0 and postoperative day (POD) 30. Follow-up included computed tomography/magnetic resonance imaging (CT/MRI), and messenger RNA (mRNA) quantification was performed by polymerase chain reaction (PCR; NETest score: 0-100; normal ≤20). Statistical analyses were performed using the Mann-Whitney U-test, Chi-square test, Kaplan-Meier survival, and area under the receiver operating characteristic curve (AUROC), as appropriate. Data are presented as mean ± standard deviation. RESULTS: The NET cohort (n = 153) included 57 patients with pancreatic cancer, 62 patients with small bowel cancer, 27 patients with lung cancer, 4 patients with duodenal cancer, and 3 patients with gastric cancer, while the surgical cohort comprised patients with R0 (n = 102) and R1 and R2 (n = 51) resection. The mean follow-up time was 14 months (range 3-68). The NETest was positive in 153/153 (100%) samples preoperatively (mean levels of 68 ± 28). In the R0 cohort, POD30 levels decreased from 62 ± 28 to 22 ± 20 (p < 0.0001), but remained elevated in 30% (31/102) of patients: 28% lung, 29% pancreas, 27% small bowel, and 33% gastric. By 18 months, 25/31 (81%) patients with a POD30 NETest >20 had image-identifiable recurrence. An NETest score of >20 predicted recurrence with 100% sensitivity and correlated with residual disease (Chi-square 17.1, p < 0.0001). AUROC analysis identified an AUC of 0.97 (p < 0.0001) for recurrence-prediction. In the R1 (n = 29) and R2 (n = 22) cohorts, the score decreased (R1: 74 ± 28 to 45 ± 24, p = 0.0012; R2: 72 ± 24 to 60 ± 28, p = non-significant). At POD30, 100% of NETest scores were elevated despite surgery (p < 0.0001). CONCLUSION: The preoperative NETest accurately identified all NETs (100%). All resections decreased NETest levels and a POD30 NETest score >20 predicted radiologically recurrent disease with 94% accuracy and 100% sensitivity. R0 resection appears to be ineffective in approximately 30% of patients. NET mRNA blood levels provide early objective genomic identification of residual disease and may facilitate management.

Circulating MicroRNAs in Small-bowel Neuroendocrine Tumors: A Potential Tool for Diagnosis and Assessment of Effectiveness of Surgical Resection.

OBJECTIVE: To discover serum-based microRNA (miRNA) biomarkers for small-bowel neuroendocrine tumors (SBNET) to help guide clinical decisions. BACKGROUND: MiRNAs are small noncoding RNA molecules implicated in the initiation and progression of many cancers. MiRNAs are remarkably stable in bodily fluids, and can potentially be translated into clinically useful biomarkers. Novel biomarkers are needed in SBNET to determine disease aggressiveness, select patients for treatment, detect early recurrence, and monitor response. METHODS: This study was performed in 3 stages (discovery, validation, and a prospective, longitudinal assessment). Discovery comprised of global profiling of 376 miRNA in sera from SBNET patients (n = 11) versus healthy controls (HCs; n = 3). Up-regulated miRNAs were subsequently validated in additional SBNET (n = 33) and HC sera (n = 14); and then longitudinally after SBNET resection (n = 12), with serial serum sampling (preoperatively day 0; postoperatively at 1 week, 1 month, and 12 months). RESULTS: Four serum miRNAs (miR-125b-5p, -362-5p, -425-5p and -500a-5p) were significantly up-regulated in SBNET (P < 0.05; fold-change >2) based on multiple normalization strategies, and were validated by RT-qPCR. This combination was able to differentiate SBNET from HC with an area under the curve of 0.951. Longitudinal assessment revealed that miR-125b-5p returned towards HC levels at 1 month postoperatively in patients without disease, whereas remaining up-regulated in those with residual disease (RSD). This was also true at 12 months postoperatively. In addition, miR-362-5p appeared up-regulated at 12 months in RSD and recurrent disease (RCD). CONCLUSIONS: Our study represents the largest global profiling of serum miRNAs in SBNET patients, and the first to evaluate ongoing serum miRNA expression changes after surgical resection. Serum miR-125b-5p and miR-362-5p have potential to be used to detect RSD/RCD.

Prospective Evaluation of the NETest as a Liquid Biopsy for Gastroenteropancreatic and Bronchopulmonary Neuroendocrine Tumors: An ENETS Center of Excellence Experience.

BACKGROUND: There is a substantial unmet clinical need for an accurate and effective blood biomarker for neuroendocrine neoplasms (NEN). We therefore evaluated, under real-world conditions in an ENETS Center of Excellence (CoE), the clinical utility of the NETest as a liquid biopsy and compared its utility with chromogranin A (CgA) measurement. METHODS: The cohorts were: gastroenteropancreatic NEN (GEP-NEN; n = 253), bronchopulmonary NEN (BPNEN; n = 64), thymic NEN (n = 1), colon cancer (n = 37), non-small-cell lung cancer (NSCLC; n = 63), benign lung disease (n = 59), and controls (n = 86). In the GEPNEN group, 164 (65%) had image-positive disease (IPD, n = 135) or were image-negative but resection-margin/biopsy-positive (n = 29), and were graded as G1 (n = 106), G2 (n = 49), G3 (n = 7), or no data (n = 2). The remainder (n = 71) had no evidence of disease (NED). In the BPNEN group, 43/64 (67%) had IPD. Histology revealed typical carcinoids (TC, n = 14), atypical carcinoids (AC, n = 14), small-cell lung cancer (SCLC, n = 11), and large-cell neuroendocrine carcinoma (LCNEC, n = 4). Disease status (stable or progressive) was evaluated according to RECIST v1.1. Blood sampling involved NETest (n = 563) and NETest/CgA analysis matched samples (n = 178). NETest was performed by PCR (on a scale of 0-100), with a score ≥20 reflecting a disease-positive status and >40 reflecting progressive disease. CgA positivity was determined by ELISA. Samples were deidentified and measurements blinded. The Kruskal-Wallis, Mann-Whitney U, and McNemar tests, and the area under the curve (AUC) of the receiver-operating characteristics (ROC) were used in the statistical analysis. RESULTS: In the GEPNEN group, NETest was significantly higher (34.4 ± 1.8, p < 0.0001) in disease-positive patients than in patients with NED (10.5 ± 1, p < 0.0001), colon cancer patients (18 ± 4, p < 0.0004), and controls (7 ± 0.5, p < 0.0001). Sensitivity for detecting disease compared to controls was 89% and specificity was 94%. NETest levels were increased in G2 vs. G1 (39 ± 3 vs. 32 ± 2, p = 0.02) and correlated with stage (localized: 26 ± 2 vs. regional/distant: 40 ± 3, p = 0.0002) and progression (55 ± 5 vs. 34 ± 2 in stable disease, p = 0.0005). In the BPNEN group, diagnostic sensitivity was 100% and levels were significantly higher in patients with bronchopulmonary carcinoids (BPC; 30 ± 1.3) who had IPD than in controls (7 ± 0.5, p < 0.0001), patients with NED (24.1 ± 1.3, p < 0.005), and NSCLC patients (17 ± 3, p = 0.0001). NETest levels were higher in patients with poorly differentiated BPNEN (LCNEC + SCLC; 59 ± 7) than in those with BPC (30 ± 1.3, p = 0.0005) or progressive disease (57.8 ± 7), compared to those with stable disease (29.4 ± 1, p < 0.0001). The AUC for differentiating disease from controls was 0.87 in the GEPNEN group and 0.99 in BPC patients (p < 0.0001). Matched CgA analysis was performed in 178 patients. In the GEPNEN group (n = 135), NETest was significantly more accurate for detecting disease (99%) than CgA positivity (53%; McNemar test χ2 = 87, p < 0.0001). In the BPNEN group (n = 43), NETest was significantly more accurate for disease detection (100%) than CgA positivity (26%; McNemar's test χ2 = 30, p < 0.0001). CONCLUSIONS: The NETest is an accurate diagnostic for GEPNEN and BPNEN. It exhibits tumor biology correlation with grading, staging, and progression. CgA as a biomarker is significantly less accurate than NETest. The NETest has substantial clinical utility that can facilitate patient management.

NETest is superior to chromogranin A in neuroendocrine neoplasia: a prospective ENETS CoE analysis.

INTRODUCTION: The absence of a reliable, universal biomarker is a significant limitation in neuroendocrine neoplasia (NEN) management. We prospectively evaluated two CgA assays, (NEOLISA, EuroDiagnostica) and (CgA ELISA, Demeditec Diagnostics (DD)) and compared the results to the NETest. METHODS: NEN cohort (n = 258): pancreatic, n = 67; small intestine, n = 40; appendiceal, n = 10; rectal, n = 45; duodenal, n = 9; gastric, n = 44; lung, n = 43. Image-positive disease (IPD) (n = 123), image & histology- negative (IND) (n = 106), and image-negative and histology positive (n = 29). CgA metrics: NEOLISA, ULN: 108 ng/mL, DD: ULN: 99 ng/mL. Data mean ± s.e.m. NETest: qRT-PCR - multianalyte analyses, ULN: 20. All samples de-identified and assessed blinded. Statistics: Mann-Whitney U-test, Pearson correlation and McNemar-test. RESULTS: CgA positive in 53/258 (NEOLISA), 32 (DD) and NETest-positive in 157/258. In image- positive disease (IPD, n = 123), NEOLISA-positive: 33% and DD: 19%. NETest-positive: 122/123 (99%; McNemar's Chi2= 79-97, P < 0.0001). NEOLISA was more accurate than DD (P = 0.0003). In image- negative disease (IND), CgA was NEOLISA-positive (11%), DD (8%), P = NS, and NETest (33%). CgA assays could not distinguish progressive (PD) from stable disease (SD) or localized from metastatic disease (MD). NETest was significantly higher in PD (47 ± 5) than SD (29 ± 1, P = 0.0009). NETest levels in MD (35 ± 2) were elevated vs localized disease (24 ± 1.3, P = 0.008). CONCLUSIONS: NETest, a multigenomic mRNA biomarker, was ~99% accurate in the identification of NEN disease. The CgA assays detected NEN disease in 19-33%. Multigenomic blood analysis using NETest is more accurate than CgA and should be considered the biomarker standard of care.

Evaluation of circulating transcript analysis (NETest) in small intestinal neuroendocrine neoplasms after surgical resection.

PURPOSE: Surgical resection is the only effective curative strategy for small intestinal neuroendocrine neoplasms (SINENs). Nevertheless, the evaluation of residual disease and prediction of disease recurrence/progression remains a problematic issue. METHODS: We evaluated 13 SINENs that underwent surgical resection of the primary tumour and/or mesenteric mass. Patients were divided in three groups: (a) Group 1: SINENs that underwent resection with curative intent, (b) Group 2: SINENs treated with resection in the setting of metastatic disease, which remained stable and (c) Group 3: SINENs treated with resection in the setting of metastatic disease, with evidence of any progression at follow-up. NETest and chromogranin A were measured pre-operatively and post-operatively during a 22-month median follow-up period and compared with imaging studies. NETest score <20% was determined as normal, 20-40% low, 41-79% intermediate and ≥80% high score. RESULTS: NETest score was raised in all (100%) SINENs pre-operatively. Surgery with curative intent resulted in NETest score reduction from 78.25 ± 15.32 to 25.25 ± 1.75 (p < 0.05). Low NETest scores post-operatively were evident in all cases without clinical evidence of residual disease (Group 1). However, the low disease activity score suggested the presence of microscopic residual disease. In three cases (75%) with stable disease (Group 2) the NETest score was low consistent with indolent disease. In the progressive disease group (Group 3), a high NETest score was present in three cases (60%) and an intermediate NETest score in the remainder (40%). CONCLUSIONS: Blood NETest scores accurately identified SINENs and were significantly decreased by curative surgery. Monitoring NETest post-operatively may facilitate management by identifying the presence of residual/progressive disease.

Molecular identification of bronchopulmonary neuroendocrine tumours and neuroendocrine genotype in lung neoplasia using the NETest liquid biopsy.

OBJECTIVES: Diagnosing lung neuroendocrine neoplasia (NEN) requires a biopsy or an operation. We evaluated a 'liquid biopsy' (NETest) as an in vitro diagnostic tool for identifying NEN and compared it to chromogranin A (CgA). METHODS: We identified 4 study cohorts: patients with bronchopulmonary carcinoids (n = 99, including 62 typical and 37 atypical carcinoids), lung cancers [n = 101, including 41 adenocarcinomas, 37 squamous carcinomas (SQC), 16 small-cell lung cancers and 7 large-cell neuroendocrine carcinomas]; benign disease (50 idiopathic pulmonary fibrosis) and healthy controls (n = 102). Transcript levels measured quantitatively (activity scores: 0-100) were compared to CgA (enzyme-linked immunosorbent assay; normal < 109 ng/ml) levels. RESULTS: The results of the NETest were positive (>20) in 94% of patients with bronchopulmonary carcinoid compared to 8% of the controls (Fisher's exact test; P < 0.001) and were significantly more accurate as a diagnostic test (McNemar's test; P < 0.001, χ2 = 72) than was CgA (positive: 19% bronchopulmonary carcinoid, 15% controls). Small-cell lung cancers (87%), large-cell neuroendocrine carcinomas (86%), adenocarcinoma (42%) and SQC (35%) were also NETest-positive. Increasing the NETest cut-off score to >40 was useful for detecting all NENs and differentiating these tumours from either controls/benign lung diseases (specificity 97%) or adenocarcinoma/SQC (specificity 94%). CgA was positive in 15-44% irrespective of pathology and had no diagnostic value. CONCLUSIONS: A gene-based liquid biopsy is an effective and accurate method for diagnosing lung tumours with neuroendocrine gene expression. CgA has no value. An NETest score >40 provides an accurate (94-97%) rule-in for the diagnosis of NEN and a rule-out for benign and other neoplastic diseases. Because neuroendocrine gene expression is associated with a poor prognosis, NETest levels may have utility both in the diagnosis of and the treatment stratification for lung neoplasia.

An Assessment of Circulating Chromogranin A as a Biomarker of Bronchopulmonary Neuroendocrine Neoplasia: A Systematic Review and Meta-Analysis.

BACKGROUND: Management of bronchopulmonary neuroendocrine neoplasia (NEN; pulmonary carcinoids [PCs], small-cell lung cancer [SCLC], and large cell neuroendocrine carcinoma) is hampered by the paucity of biomarkers. Chromogranin A (CgA), the default neuroendocrine tumor biomarker, has undergone wide assessment in gastroenteropancreatic neuroendocrine tumors. OBJECTIVES: To evaluate CgA in lung NEN, define its clinical utility as a biomarker, assess its diagnostic, prognostic, and predictive efficacy, as well as its accuracy in the identification of disease recurrence. METHODS: A systematic review of PubMed was undertaken using the preferred reporting items for systematic reviews and meta-analyses guidelines. No language restrictions were applied. Overall, 33 original scientific papers and 3 case reports, which met inclusion criteria, were included in qualitative analysis, and meta-analysis thereafter. All studies, except 2, were retrospective. Meta-analysis statistical assessment by generic inverse variance methodology. RESULTS: Ten different CgA assay types were reported, without consistency in the upper limit of normal (ULN). For PCs (n = 16 studies; median patient inclusion 21 [range 1-200, total: 591 patients]), the CgA diagnostic sensitivity was 34.5 ± 2.7% with a specificity of 93.8 ± 4.7. CgA metrics were not available separately for typical or atypical carcinoids. CgA >100 ng/mL (2.7 × ULN) and >600 ng/mL (ULN unspecified) were anecdotally prognostic for overall survival (n = 2 retrospective studies). No evidence was presented for predicting treatment response or identifying post-surgery residual disease. For SCLC (n = 19 studies; median patient inclusion 23 [range 5-251, total: 1,241 patients]), the mean diagnostic sensitivity was 59.9 ± 6.8% and specificity 79.4 ± 3.1. Extensive disease typically exhibited higher CgA levels (diagnostic accuracy: 61 ± 2.5%). An elevated CgA was prognostic for overall survival (n = 4 retrospective studies). No prospective studies evaluating predictive benefit or prognostic utility were identified. CONCLUSION: The available data are scarce. An assessment of all published data showed that CgA exhibits major limitations as an effective and accurate biomarker for either PC or SCLC. Its utility especially for localized PC/limited SCLC (when surgery is potentially curative), is limited. The clinical value of CgA remains to be determined. This requires validated, well-constructed, multicenter, prospective, randomized studies. An assessment of all published data indicates that CgA does not exhibit the minimum required metrics to function as a clinically useful biomarker for lung NENs.

Blood Chromogranin A Is Not Effective as a Biomarker for Diagnosis or Management of Bronchopulmonary Neuroendocrine Tumors/Neoplasms.

BACKGROUND: Identification of circulating tumor markers for clinical management in bronchopulmonary (BP) neuroendocrine tumors/neoplasms (NET/NEN) is of considerable clinical interest. Chromogranin A (CgA), a "universal" NET biomarker, is considered controversial as a circulating biomarker of BPNEN. AIM: Assess utility of CgA in the diagnosis and management of BPNEN in a multicentric study. MATERIAL AND METHODS: CgA diagnostic metrics were assessed in lung NET/NENs (n = 200) and controls (n = 140), randomly assigned to a Training and Test set (100 BPC and 70 controls in each). Assay specificity was evaluated in neoplastic lung disease (n = 137) and nonneoplastic lung disease (n = 77). CgA efficacy in predicting clinical status was evaluated in the combined set of 200 NET/NENs. CgA levels in bronchopulmonary neuroendocrine tumor (BPNET) subtypes (atypical [AC] vs. typical [TC]) and grade was examined. The clinical utility of an alteration of CgA levels (±25%) was evaluated in a subset of 49 BPNET over 12 months. CgA measurement was by NEOLISATM kit (EuroDiagnostica). RESULTS: Sensitivity and specificity in the training set were 41/98%, respectively. Test set data were 42/87%. Training set area under receiver operator characteristic analysis differentiated BPC from control area under the curve (AUC) 0.61 ± 0.05 p = 0.015. Test set the data were AUC 0.58 ± 0.05, p = 0.076. In the combined set (n = 200), 67% BPNET/NEN (n = 134) had normal CgA levels. CgA levels did not distinguish histological subtypes (TC vs. AC, AUC 0.56 ± 0.04, p = 0.21), grade (p = 0.45-0.72), or progressive from stable disease (AUC 0.53 ± 0.05 p = 0.47). There was no correlation of CgA with Ki-67 index (Pearson r = 0.143, p = 0.14). For nonneoplastic diseases (chronic obstructive pulmonary disorder and idiopathic pulmonary fibrosis), CgA was elevated in 26-37%. For neoplastic disease (NSCLC, squamous cell carcinoma), CgA was elevated in 11-16%. The neuroendocrine SCLC also exhibited elevated CgA (50%). Elevated CgA was not useful for differentiating BPNET/NEN from these other pathologies. Monitoring BPNET/NEN over a 12-month period identified neither CgA levels per se nor changes in CgA were reflective of somatostatin analog treatment outcome/efficacy or the natural history of the disease (progression). CONCLUSIONS: Blood CgA levels are not clinically useful as a biomarker for lung BPNET/NEN. The low specificity and elevations in both nonneoplastic as well as other common neoplastic lung diseases identified limited clinical utility for this biomarker.

The clinical applications of a multigene liquid biopsy (NETest) in neuroendocrine tumors.

PURPOSE: There are few effective biomarkers for neuroendocrine tumors. Precision oncology strategies have provided liquid biopsies for real-time and tailored decision-making. This has led to the development of the first neuroendocrine tumor liquid biopsy (the NETest). The NETest represents a transcriptomic signature of neuroendocrine tumor (NETs) that captures tumor biology and disease activity. The data have direct clinical application in terms of identifying residual disease, disease progress and the efficacy of treatment. In this overview we assess the available published information on the metrics and clinical efficacy of the NETest. MATERIAL AND METHODS: Published data on the NETest have been collated and analyzed to understand the clinical application of this multianalyte biomarker in NETs. RESULTS: NETest assay has been validated as a standardized and reproducible clinical laboratory measurement. It is not affected by demographic characteristics, or acid suppressive medication. Clinical utility of the NETest has been documented in gastroenteropancreatic, bronchopulmonary NETs, in paragangliomas and pheochromocytomas. The test facilitates accurate diagnosis of a NET disease, and real-time monitoring of the disease status (stable/progressive disease). It predicts aggressive tumor behavior, identifies operative tumor resection, and efficacy of the medical treatment (e.g. somatostatin analogues), or peptide receptor radionuclide therapy (PRRT). NETest metrics and clinical applications out-perform standard biomarkers like chromogranin A. CONCLUSIONS: The NETest exhibits clinically competent metrics as an effective biomarker for neuroendocrine tumors. Measurement of NET transcripts in blood is a significant advance in neuroendocrine tumor management and demonstrates that blood provides a viable source to identify and monitor tumor status.

PRRT neuroendocrine tumor response monitored using circulating transcript analysis: the NETest.

PURPOSE: Peptide receptor radionuclide therapy (PRRT) is effective for metastatic/inoperable neuroendocrine tumors (NETs). Imaging response assessment is usually efficient subsequent to treatment completion. Blood biomarkers such as PRRT Predictive Quotient (PPQ) and NETest are effective in real-time. PPQ predicts PRRT efficacy; NETest monitors disease. We prospectively evaluated: (1) NETest as a surrogate biomarker for RECIST; (2) the correlation of NETest levels with PPQ prediction. METHODS: Three independent 177Lu-PRRT-treated GEP-NET and lung cohorts (Meldola, Italy: n = 72; Bad-Berka, Germany: n = 44; Rotterdam, Netherlands: n = 41). Treatment response: RECIST1.1 (responder (stable, partial, and complete response) vs non-responder). Blood sampling: pre-PRRT, before each cycle and follow-up (2-12 months). PPQ (positive/negative) and NETest (0-100 score) by PCR. Stable < 40; progressive > 40). CgA (ELISA) as comparator. Samples de-identified, measurement and analyses blinded. Kaplan-Meier survival and standard statistics. RESULTS: One hundred twenty-two of the 157 were evaluable. RECIST stabilization or response in 67%; 33% progressed. NETest significantly (p < 0.0001) decreased in RECIST "responders" (- 47 ± 3%); in "non-responders," it remained increased (+ 79 ± 19%) (p < 0.0005). NETest monitoring accuracy was 98% (119/122). Follow-up levels > 40 (progressive) vs stable (< 40) significantly correlated with mPFS (not reached vs. 10 months; HR 0.04 (95%CI, 0.02-0.07). PPQ response prediction was accurate in 118 (97%) with a 99% accurate positive and 93% accurate negative prediction. NETest significantly (p < 0.0001) decreased in PPQ-predicted responders (- 46 ± 3%) and remained elevated or increased in PPQ-predicted non-responders (+ 75 ± 19%). Follow-up NETest categories stable vs progressive significantly correlated with PPQ prediction and mPFS (not reached vs. 10 months; HR 0.06 (95%CI, 0.03-0.12). CgA did not reflect PRRT treatment: in RECIST responders decrease in 38% and in non-responders 56% (p = NS). CONCLUSIONS: PPQ predicts PRRT response in 97%. NETest accurately monitors PRRT response and is an effective surrogate marker of PRRT radiological response. NETest decrease identified responders and correlated (> 97%) with the pretreatment PPQ response predictor. CgA was non-informative.

Utility of a ready-to-use PCR system for neuroendocrine tumor diagnosis.

BACKGROUND: Multigene-based PCR tests are time-consuming and limiting aspects of the protocol include increased risk of operator-based variation. In addition, such protocols are complex to transfer and reproduce between laboratories. AIMS: Evaluate the clinical utility of a pre-spotted PCR plate (PSP) for a novel multigene (n = 51) blood-based gene expression diagnostic assay for neuroendocrine tumors (NETs). METHODS: A pilot study (n = 44; 8 controls and 36 NETs) was undertaken to compare CQ, normalized gene expression and algorithm-based output (NETest score). Gene expression was then evaluated between matched blood:tumor tissue samples (n = 7). Thereafter, two prospective sets (diagnostic: n = 167; clinical validation: n = 48, respectively) were evaluated for diagnostic and clinical utility value. Two independent molecular diagnostics facilities were used to assess assay reproducibility and inter-laboratory metrics. Samples were collected (per CLIA protocol) processed to mRNA and cDNA and then either run per standard assay (liquid primers) or on PSPs. Separately, matching plasma samples were analyzed for chromogranin A (CgA). Statistics included non-parametric testing, Pearson-concordance, Predictive Modeling and AUROC analyses. RESULTS: In the pilot study (n = 44), CQ values were highly concordant (r: 0.82, p<0.0001) and normalized gene expression data significantly related (p<0.0001) (Pearson-pairwise correlation). NETest values were not different (49.7±33 standard vs. 48.5±31.5 PSP) and the overall concordance in output 96%. Predictive modelling confirmed this concordance (F1 score = 0.95). Gene expression levels were highly correlated between blood and tumor tissue (R: 0.71-0.83). In the diagnostic cohort (n = 30 controls, n = 87 non-NET controls, n = 50 NET), NETest was significantly lower (p<0.0001) in controls (11±6.5) and non-NET controls (13±18) than NETs (61±31). The AUROCs were 0.93-0.97 and the diagnostic accuracy was 90-97.5%. As a diagnostic, the PSP-NETest was significantly better than CgA (accuracy: 56%, p<0.0001). For clinical samples, the PSP generated robust and accurate (>96%) scores and was significantly better (p<0.0001) than CgA. The assay protocol was consistent (r: 0.97) and reproducible (co-efficient of variation: 1.3-4.2%) across the two facilities. CONCLUSION: The PSP protocol for the NETest has been established and prospectively tested in clinical samples. It is highly reproducible, has similar metrics (CV, categorization by control or NET) to the standard PCR assay and generates clinically concordant (>96%) NETest results. Moreover, it functions significantly more accurately than CgA.

NETest liquid biopsy is diagnostic of small intestine and pancreatic neuroendocrine tumors and correlates with imaging.

INTRODUCTION: Current monoanalyte biomarkers are ineffective in gastroenteropancreatic neuroendocrine tumors (GEP-NETs). NETest, a novel multianalyte signature, provides molecular information relevant to disease biology. AIM(S): Independently validate NETest to diagnose GEP-NETs and identify progression in a tertiary referral center. MATERIALS AND METHODS: Cohorts: 67 pancreatic NET (PNETs), 44 small intestine NETs (SINETs), 63 controls. Well-differentiated (WD): PNETs, n=62, SINETs, all (n=44). Disease extent assessment at blood draw: anatomical (n=110)- CT(n=106), MRI(n=7) and/or functional- 68Ga-SSA-PET/CT(n=69) or 18F-FDG-PET/CT (n=8). Image positive disease (IPD) was defined as either CT/MRI or 68Ga-SSA-PET/CT/18F-FDG-PET/CT-positive. Both CT/MRI and 68Ga-SSA-PET/CT-negative in WD-NETs was considered image negative disease (IND). NETest (normal: 20): PCR (spotted plates). DATA: mean±SD. RESULTS: Diagnosis: NETest was significantly increased in NETs (n=111; 26±21) vs. controls (8±4, p<0.0001). 75 (42 PNET, 33 SINET) were image-positive. Eleven (8 PNET, 3 SINET; all WD) were IND. In IPD, NETest was significantly higher (36±22) vs. IND (8±7, p<0.0001). NETest accuracy, sensitivity, specificity: 97%, 99%, 95%. Concordance with imaging: NETest was 92% (101/110) concordant with anatomical imaging, 94% (65/69) with 68Ga-SSA-PET/CT, 96% (65/68) dual modality (CT/MRI and 68Ga-SSA-PET/CT). In 70 CT/MRI-positive, NETest was elevated in all (37±22). In 40 CT/MRI-negative, NETest was normal (11±10) in 31. In 56 68Ga-SSA-PET/CT-positive, NETest was elevated (36±22) in 55. In 13 68Ga-SSA-PET/CT-negative, NETest was normal (9±8) in 10. Disease status: NETest was significantly higher in progressive (61±26; n=11) vs. stable disease (29±14; n=64; p<0.0001) (RECIST 1.1). CONCLUSION: NETest is an effective diagnostic for PNETs and SINETs. Elevated NETest is as effective as imaging in diagnosis and accurately identifies progression.