Cell-free DNA methylation analysis as a marker of malignancy in pleural fluid.

Diagnosis of malignant pleural effusion (MPE) is made by cytological examination of pleural fluid or histological examination of pleural tissue from biopsy. Unfortunately, detection of malignancy using cytology has an overall sensitivity of 50%, and is dependent upon tumor load, volume of fluid assessed, and cytopathologist experience. The diagnostic yield of pleural fluid cytology is also compromised by low abundance of tumor cells or when morphology is obscured by inflammation or reactive mesothelial cells. A reliable molecular marker that may complement fluid cytology for the diagnosis of malignant pleural effusion is needed. The purpose of this study was to establish a molecular diagnostic approach based on pleural effusion cell-free DNA methylation analysis for the differential diagnosis of malignant pleural effusion and benign pleural effusion. This was a blind, prospective case-control biomarker study. We recruited 104 patients with pleural effusion for the study. We collected pleural fluid from patients with: MPE (n = 48), indeterminate pleural effusion in subjects with known malignancy or IPE (n = 28), and benign PE (n = 28), and performed the Sentinel-MPE liquid biopsy assay. The methylation level of Sentinel-MPE was markedly higher in the MPE samples compared to BPE control samples (p < 0.0001) and the same tendency was observed relative to IPE (p = 0.004). We also noted that the methylation signal was significantly higher in IPE relative to BPE (p < 0.001). We also assessed the diagnostic efficiency of the Sentinel-MPE test by performing receiver operating characteristic analysis (ROC). For the ROC analysis we combined the malignant and indeterminate pleural effusion groups (n = 76) and compared against the benign group (n = 28). The detection sensitivity and specificity of the Sentinel-MPE test was high (AUC = 0.912). The Sentinel-MPE appears to have better performance characteristics than cytology analysis. However, combining Sentinel-MPE with cytology analysis could be an even more effective approach for the diagnosis of MPE. The Sentinel-MPE test can discriminate between BPE and MPE. The Sentinel-MPE liquid biopsy test can detect aberrant DNA in several different tumor types. The Sentinel-MPE test can be a complementary tool to cytology in the diagnosis of MPE.

Cell-Free DNA Methylation Analysis as a Marker of Malignancy in Pleural Fluid.

Background: Diagnosis of malignant pleural effusion (MPE) is made by cytological examination of pleural fluid or histological examination of pleural tissue from biopsy. Unfortunately, detection of malignancy using cytology has an overall sensitivity of 50%, and is dependent upon tumor load, volume of fluid assessed, and cytopathologist experience. The diagnostic yield of pleural fluid cytology is also compromised by low abundance of tumor cells or when morphology is obscured by inflammation or reactive mesothelial cells. A reliable molecular marker that may complement fluid cytology malignant pleural effusion diagnosis is needed. The purpose of this study was to establish a molecular diagnostic approach based on pleural effusion cell-free DNA methylation analysis for the differential diagnosis of malignant pleural effusion and benign pleural effusion. Results: This was a blind, prospective case-control biomarker study. We recruited 104 patients with pleural effusion for the study. We collected pleural fluid from patients with: MPE (n = 48), PPE (n = 28), and benign PE (n = 28), and performed the Sentinel-MPE liquid biopsy assay. The methylation level of Sentinel-MPE was markedly higher in the MPE samples compared to BPE control samples (p < 0.0001) and the same tendency was observed relative to PPE (p = 0.004). We also noted that the methylation signal was significantly higher in PPE relative to BPE (p < 0.001). We also assessed the diagnostic efficiency of the Sentinel-MPE test by performing receiver operating characteristic analysis (ROC). For the ROC analysis we combined the malignant and paramalignant groups (n = 76) and compared against the benign group (n = 28). The detection sensitivity and specificity of the Sentinel-MPE test was high (AUC = 0.912). The Sentinel-MPE appears to have better performance characteristics than cytology analysis. However, combining Sentinel-MPE with cytology analysis could be an even more effective approach for the diagnosis of MPE. Conclusions: The Sentinel-MPE test can discriminate between BPE and MPE. The Sentinel-MPE liquid biopsy test can detect aberrant DNA in several different tumor types. The Sentinel-MPE test can be a complementary tool to cytology in the diagnosis of MPE.

A Comprehensive Evaluation of Special AT-rich Sequence-binding Protein 2 (SATB2) Immunohistochemical Staining in Mucinous Tumors From Gastrointestinal and Nongastrointestinal Sites.

Special AT-rich sequence-binding protein 2 (SATB2) is an accurate marker for conventional colorectal carcinoma (CRC), although its sensitivity and specificity in mucinous tumors from the colon and other sites remains unknown. The objective of this study is to evaluate the accuracy of SATB2 expression detected by immunohistochemical assay, as a marker of primary CRC in mucinous adenocarcinomas. SATB2 immunohistochemical stains were performed on whole sections from 63 conventional CRCs (controls), 47 mucinous CRCs (mCRC), and 182 noncolorectal mucinous tumors. SATB2 intensity was scored as 1 to 3 based on the estrogen receptor/progesterone receptor grading system, and the percent positive cells was scored in broad categories as follows: 0 (negative)≤5%, 1=5% to 49%, 2≥50%. An optimal sensitivity/specificity pairing (83% and 95%, respectively) was achieved in the mCRCs when the additive intensity and percent score was ≥3 (ie, intensity score+percent score=total score). Defining this total score (histologic score/"H score") as a "positive" result, the sensitivity of SATB2 for conventional CRC was 98% (62/63) versus 83% (39/47) for mCRCs (P=0.02); whereas 5% (9/182) of all noncolorectal mucinous tumors were considered positive. SATB2 especially demonstrated reduced specificity when applied to mucinous gastroesophageal and breast carcinomas, which showed significant expression in 27% and 9% of cases, respectively. In summary, SATB2 is a less sensitive marker of colorectal origin in mCRC compared with conventional CRC and shows significantly reduced specificity in mucinous gastroesophageal and breast primaries.

Selective staining of gastric biopsies for H. pylori does not affect detection rates or turnaround time and improves cost compared to reflexive staining.

AIMS: To evaluate how reflexive versus selective H. pylori stains affect detection rates, turnaround time (TAT), and cost savings in a real life practice environment following an institutional policy change. METHODS: The aforementioned parameters were evaluated in all cases in the year preceding and the year following an institutional policy change from reflexive to selective staining. RESULTS: 1497 patients comprised the reflexive stain (RS) group of which 228 (15.2%) were H. pylori positive. 1629 patients comprised the selective stain (SS) group of which 237 (14.5%) were H. pylori positive. There was no significant difference in H. pylori detection rates between the RS and SS groups (OR=0.95, 95% CI=0.78-1.15, p=0.59). TATs were similarly equivalent with a mean of 52.4h for the RS cohort and 53.7h for the SS cohort (p=0.344), both of which included a resident preview day. We calculated an average laboratory cost savings of $11.68 per case, which saved our department over $15,000 (37%) in the year following the policy change. CONCLUSIONS: Our results support a policy of selective staining for H. pylori as opposed to reflexive staining and go on to show that laboratories that change their policy can expect to generate cost savings without compromising detection rates or TAT.