BACKGROUND:The authors previously developed an artificial intelligence (AI) to assist cytologists in the evaluation of digital whole-slide images (WSIs) generated from bile duct brushing specimens. The aim of this trial was to assess the efficiency and accuracy of cytologists using a novel application with this AI tool. METHODS:Consecutive bile duct brushing WSIs from indeterminate strictures were obtained. A multidisciplinary panel reviewed all relevant information and provided a central interpretation for each WSI as being "positive," "negative," or "indeterminate." The WSIs were then uploaded to the AI application. The AI scored each WSI as positive or negative for malignancy (i.e., computer-aided diagnosis [CADx]). For each WSI, the AI prioritized cytologic tiles by the likelihood that malignant material was present in the tile. Via the AI, blinded cytologists reviewed all WSIs and provided interpretations (i.e., computer-aided detection [CADe]). The diagnostic accuracies of the WSI evaluation via CADx, CADe, and the original clinical cytologic interpretation (official cytologic interpretation [OCI]) were compared. RESULTS:Of the 84 WSIs, 15 were positive, 42 were negative, and 27 were indeterminate after central review. The WSIs generated on average 141,950 tiles each. Cytologists using the AI evaluated 10.5 tiles per WSI before making an interpretation. Additionally, cytologists required an average of 84.1 s of total WSI evaluation. WSI interpretation accuracies for CADx (0.754; 95% CI, 0.622-0.859), CADe (0.807; 95% CI, 0.750-0.856), and OCI (0.807; 95% CI, 0.671-0.900) were similar. CONCLUSIONS:This trial demonstrates that an AI application allows cytologists to perform a triaged review of WSIs while maintaining accuracy.

University of Massachusetts Medical School, USA; Mayo Clinic Minnesota, USA; Allina Health, USA.

PDF file, 83KB, Biopsy sites of validation study cases.

Objective. Alterations in DNA methylation are early events in endometrial cancer (EC) development and may have utility in EC detection via tampon-collected vaginal fluid. Methods. For discovery, DNA from frozen EC, benign endometrium (BE), and benign cervicovaginal (BCV) tissues underwent reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs). Candidate DMRs were selected based on receiver operating characteristic (ROC) discrimination, methylation level fold-change between cancers and controls, and absence of background CpG methylation. Methylated DNA marker (MDM) validation was performed using qMSP on DNA from independent EC and BE FFPE tissue sets. Women >= 45 years of age with abnormal uterine bleeding (AUB) or postmenopausal bleeding (PMB) or any age with biopsy-proven EC self-collected vaginal fluid using a tampon prior to clinically indicated endometrial sampling or hysterectomy. Vaginal fluid DNA was assayed by qMSP for EC-associated MDMs. Random forest modeling analysis was performed to generate predictive probability of underlying disease; results were 500-fold in-silico cross-validated. Results. Thirty-three candidate MDMs met performance criteria in tissue. For the tampon pilot, 100 EC cases were frequency matched by menopausal status and tampon collection date to 92 BE controls. A 28-MDM panel highly discriminated between EC and BE (96% (95%CI 89-99%) specificity; 76% (66-84%) sensitivity (AUC 0.88). In PBS/EDTA tampon buffer, the panel yielded 96% (95% CI 87-99%) specificity and 82% (70-91%) sensitivity (AUC 0.91).

PDF file, 85KB, Supplemental Table 1: Performance characteristics of the 92-gene assay for tumor subclassification. Supplemental Table 2: Pre-analytical variables and effects on 92-gene assay performance

University of Massachusetts Medical School, USA; Mayo Clinic Minnesota, USA; Allina Health, USA.

Context.-The US Food and Drug Administration (FDA) approved immune checkpoint inhibitor therapy for pa-tients with advanced solid tumors that have DNA mismatch repair defects or high levels of microsatellite instability; however, the FDA provided no guidance on which specific clinical assays should be used to determine mismatch repair status.Objective.-To develop an evidence-based guideline to identify the optimal clinical laboratory test to identify defects in DNA mismatch repair in patients with solid tumor malignancies who are being considered for immune checkpoint inhibitor therapy.Design.-The College of American Pathologists con-vened an expert panel to perform a systematic review of the literature and develop recommendations. Using the National Academy of Medicine-endorsed Grading of Recommendations Assessment, Development and Evalua-tion approach, the recommendations were derived from available evidence, strength of that evidence, open comment feedback, and expert panel consensus. Mismatch repair immunohistochemistry, microsatellite instability derived from both polymerase chain reaction and next -generation sequencing, and tumor mutation burden derived from large panel next-generation sequencing were within scope.Results.-Six recommendations and 3 good practice statements were developed. More evidence and evidence of higher quality were identified for colorectal cancer and other cancers of the gastrointestinal (GI) tract than for cancers arising outside the GI tract.Conclusions.-An optimal assay depends on cancer type. For most cancer types outside of the GI tract and the endometrium, there was insufficient published evidence to recommend a specific clinical assay. Absent published evidence, immunohistochemistry is an acceptable ap-proach readily available in most clinical laboratories.

Objectives DNA methylation occurs early in carcinogenesis and differ among cancer subtypes, suggest that these biomarkers might be useful in early detection and in defining site of origin in liquid biopsies. Methods Endometrial (EC), ovarian (OC) and cervical (CC) cancer MDMs were identified through methylome sequencing of primary tumor and benign gynecologic tissue DNA and independently validated in DNA from formalin fixed paraffin embedded (FFPE) tissues representing the same cancers and benign histologies. Twenty-five MDMs representing each organ site were selected as previously reported; DNA extracted from independent primary tissues was assayed by quantitative methylation specific PCR. MDMs were normalized to β-actin. MDM distributions were displayed using boxplots and intensity maps. Results 82 EC (16 serous, 18 carcinosarcoma, 7 clear cell, 17 endometrioid grade 1/2, 24 endometrioid grade 3), 82 OC (36 serous, 21 clear cell, 4 mucinous, 21 endometrioid), and 64 CC (36 squamous cell, 28 adenocarcinoma) were compared to controls of benign epithelium (29 cervicovaginal, 29 fallopian tube, 14 benign endometrial tissues). While CDO1 discriminated any cancer type from benign control tissue, cancer specificity was evident for most MDMs (figure 1). Overlap of MDMs, such as c18orf18 among EC and OC clear cell and endometrioid histologies, is compatible with the origin of these OCs from endometriosis (figure 2). Conclusions MDMs discovered and independently validated in EC, OC, and CC tissues discriminate among GC site origin. MDM testing in vaginal fluid and/or blood is warranted to assess GC detection and site-specificity via non-invasive liquid biopsy.

Neil Marya: YES financial relationship with a commercial interest;Boston Scientific:Consulting | Christopher Hartley: NO financial relationship with a commercial interest | Patrick Powers: NO financial relationship with a commercial interest | Melanie Bois: NO financial relationship with a commercial interest | Sarah Kerr: NO financial relationship with a commercial interest | Ferga Gleeson: NO financial relationship with a commercial interest | Barham Abu Dayyeh: YES financial relationship with a commercial interest;Boston Scientific:Consulting;Medtronic :Consulting;Endogenex:Advisory Committees or Review Panels;Apollo Endosurgery :Grant/Research Support;USGI Medical :Grant/Research Support;Nitinotes :Advisory Committees or Review Panels;Hemostasis :Consulting;BFKW:Consulting;EndoTagss:Consulting;Metamodix :Consulting;Olympus Speaking and Teaching;Johnson and Johnson:Speaking and Teaching | Benjamin Kipp: NO financial relationship with a commercial interest | Ryan Law: YES financial relationship with a commercial interest;Conmed:Consulting;Medtronic:Consulting | John Martin: NO financial relationship with a commercial interest | Bret Petersen: YES financial relationship with a commercial interest;Olympus:Advisory Committees or Review Panels;Boston Scientific Corp:Grant/Research Support;AMBU:Grant/Research Support;Pentax:Consulting;Abbvie:Stock Shareholder;Pfizer:Consulting;Pfizer:Stock Shareholder;Moderna:Stock Shareholder;Abbott:Stock Shareholder;Merck:Stock Shareholder;Editas Medicine:Stock Shareholder | Andrew Storm: YES financial relationship with a commercial interest;Apollo Endosurgery:Grant/Research Support;Boston Scientific:Grant/Research Support;ERBE:Consulting;Endo-TAGSS:Grant/Research Support;Endogenex:Grant/Research Support;Enterasense:Grant/Research Support;Olympus:Consulting | Eric Vargas: NO financial relationship with a commercial interest | Lewis Roberts: YES financial relationship with a commercial interest;Bayer:Grant/Research Support;Boston Scientific:Grant/Research Support;Exact Sciences:Grant/Research Support;Fujifilm Medical Systems:Grant/Research Support;Gilead Sciences:Grant/Research Support;Glycotest, Inc:Grant/Research Support;Redhill Biopharma:Grant/Research Support;TARGET PharmaSolutions:Grant/Research Support;MedEd Design:Consulting;Pontifax:Consulting;Global Life Science Consulting:Consulting;The Lynx Group:Consulting;Astra Zeneca:Advisory Committees or Review Panels;Bayer:Advisory Committees or Review Panels;Eisai:Advisory Committees or Review Panels;Exact Sciences:Advisory Committees or Review Panels;GRAIL:Advisory Committees or Review Panels;QED Therapeutics:Advisory Committees or Review Panels;Roche:Advisory Committees or Review Panels | Gregory Gores: NO financial relationship with a commercial interest | Rondell Graham: YES financial relationship with a commercial interest;Incyte:Advisory Committees or Review Panels;Bristol Myers Squibb:Grant/Research Support | Michael Levy: NO financial relationship with a commercial interest

5526 Background: HR-HPV DNA testing, with or without cervical cytology, provides excellent sensitivity for detection of cervical cancer (CC) and its precursors; negative test results indicate that risk of disease is extremely low and enable women to undergo reduced screening with safety. However, management of women who screen positive remains challenging as many will prove to have self-limited HR-HPV infections. DNA methylation is an early event in carcinogenesis that could enhance CC screening specificity. Methods: For discovery, DNA from 70 FFPE CC (36 squamous, 34 adenocarcinoma) tissues that were reviewed microscopically, 18 fresh frozen benign cervicovaginal (BCV) tissues collected at the time of benign hysterectomy, and 18 buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify MDMs associated with CC. Candidate MDM selection was based on area under the receiver operating characteristic curve (AUC) discrimination, methylation fold change, and low background methylation among benign controls. Candidate MDMs were re-tested using methylation-specific PCR (MSP) to confirm performance. Blinded biological validation was performed using MSP on DNA extracted from independent FFPE CC (38 squamous, 43 adenocarcinoma) and BCV (40) tissues. The performance of CC MDMs was also tested in DNA extracted from cervical dysplasia (36 adenocarcinoma in situ (AIS), 32 cervical intraepithelial neoplasia (CIN) 2/3, 11 CIN 1) FFPE tissues. Results: From RRBS discovery and technical validation via MSP, 30 candidate MDMs showed marked methylation fold changes (10 to >1000) across both CC histologies compared to BCV tissue from cancer-free women. Each of the 30 MDMs highly discriminated CC from BCV tissue with 9 MDMs having an AUC >0.90 (Table). CC MDMs also highly discriminated AIS from BCV but did not perform well in CIN 2/3 and CIN 1 (Table). Conclusions: Whole methylome sequencing, stringent filtering criteria, and biological validation have yielded outstanding candidate MDMs for CC that highly discriminate CC from BCV, notably with high specificity. Performance in cervical dysplasias varied with higher positivity rates in AIS than in CIN 2/3 and CIN 1. Translation to testing these novel MDMs in lower genital tract biospecimens and the addition of HR-HPV to the CC panel are warranted.[Table: see text]

Abstract Background Abnormal uterine bleeding requires the investigation of the endometrium. Histology is typically used but there remains room for the improvement and use of cytology. Methods Women presenting for clinically indicated office endometrial biopsy were prospectively enrolled. Tao endometrial brushing and office endometrial biopsy were performed, and surgical procedure if clinically indicated. Tao brush cytology specimens were blindly reviewed by up to three pathologists, consensus obtained, and scored as: benign, atypical (favor benign), suspicious, positive for malignancy, or non‐diagnostic. Cytology and histology were compared to surgical pathology to determine sensitivity, specificity, positive, and negative predictive values to detect AH (atypical hyperplasia) or EC (endometrial cancer). Results Clinical indications of 197 enrolled patients included postmenopausal bleeding (90, 45.7%), abnormal uterine bleeding (94, 47.7%), and abnormal endometrium on ultrasound without bleeding (13, 6.6%). Of the 197 patients, 185 (93.9%) had cytology score consensus and a total of 196 (99.5%) had consensus regarding cytology positivity. Surgical pathology diagnoses (N = 85) were 13 (15.3%) FIGO grade 1 or 2 EC, 3 (3.5%) AH, and 69 (81.2%) benign endometrium. Sensitivity and specificity to detect EC or AH were 93.7% and 100%, respectively, via endometrial biopsy; 87.5% and 63.8%, respectively, via endometrial cytology when scores of malignancy, suspicious, or atypical were considered positive. Conclusions In a high‐risk population, Tao brush endometrial cytology showed high sensitivity to detect AH and EC comparable to biopsy histology when considering scores of malignancy, suspicious, atypical, and non‐diagnostic. Revisiting the potential value of endometrial cytology in the contemporary era of endometrial diagnostic workup is warranted.

CONTEXT.—:Comprehensive genomic profiling has demonstrated that approximately 20% of pancreatic carcinomas with acinar differentiation harbor potentially targetable BRAF fusions that activate the MAPK pathway.OBJECTIVES.—:To validate the above finding by BRAF break-apart fluorescence in situ hybridization (FISH) in a large series of pure acinar cell carcinomas (ACCs), evaluate tumors for the presence of BRAF V600E mutations, and compare clinicopathologic features of tumors with BRAF rearrangements with those without.DESIGN.—:Thirty cases of pure ACC and 6 cases of mixed acinar-neuroendocrine carcinoma (ACC-NEC) were retrieved. A break-apart FISH probe was used to detect BRAF rearrangements. Immunohistochemistry for BRAF V600E was performed.RESULTS.—:BRAF rearrangements by FISH were found in 6 of 36 cases (17%), 5 of which were pure ACC and 1 was a mixed ACC-NEC. Follow-up was available in 29 of 36 cases (81%). The median survival was 22 months for BRAF-rearranged cases and 16 months for BRAF-intact cases; the 2-year overall survival was 50% for BRAF-rearranged cases and 35% for BRAF-intact cases. No significant clinicopathologic differences were identified in cases with BRAF rearrangement compared with those without BRAF rearrangement. BRAF V600E mutation was identified in 2 of 34 cases (6%), both of which were pure ACC and were BRAF-intact by FISH.CONCLUSIONS.—:This study supports the finding that BRAF rearrangements are present in approximately 20% of cases and identified BRAF V600E mutations in approximately 5% of cases. These cases may benefit from targeted therapy.

A crucial mutational mechanism in malignancy is structural variation, in which chromosomal rearrangements alter gene functions that drive cancer progression. Herein, the presence and pattern of structural variations were investigated in twelve prospectively acquired treatment‐naïve pancreatic cancers specimens obtained via endoscopic ultrasound (EUS). In many patients, this diagnostic biopsy procedure and specimen is the only opportunity to identify somatic clinically relevant actionable alterations that may impact their care and outcome. Specialized mate pair sequencing (MPseq) provided genome‐wide structural variance analysis (SVA) with a view to identifying prognostic markers and possible therapeutic targets. MPseq was successfully performed on all specimens, identifying highly rearranged genomes with complete SVA on all specimens with > 20% tumour content. SVA identified chimeric fusion proteins and potentially immunogenic readthrough transcripts, change of function truncations, gains and losses of key genes linked to tumour progression. Complex localized rearrangements, termed chromoanagenesis, with broad pattern heterogeneity were observed in 10 (83%) specimens, impacting multiple genes with diverse cellular functions that could influence theragnostic evaluation and responsiveness to immunotherapy regimens. This study indicates that genome‐wide MPseq can be successfully performed on very limited clinically EUS obtained specimens for chromosomal rearrangement detection and potential theragnostic targets.

Gestational trophoblastic disease (GTD) is a heterogeneous group of lesions arising from placental tissue. Epithelioid trophoblastic tumor (ETT), derived from chorionic-type trophoblast, is the rarest form of GTD with only approximately 130 cases described in the literature. Due to its morphologic mimicry of epithelioid smooth muscle tumors and carcinoma, ETT can be misdiagnosed. To date, molecular characterization of ETTs is lacking. Furthermore, ETT is difficult to treat when disease spreads beyond the uterus. Here using RNA-Seq analysis in a cohort of ETTs and other gestational trophoblastic lesions we describe the discovery of LPCAT1-TERT fusion transcripts that occur in ETTs and coincide with underlying genomic deletions. Through cell-growth assays we demonstrate that LPCAT1-TERT fusion proteins can positively modulate cell proliferation and therefore may represent future treatment targets. Furthermore, we demonstrate that TERT upregulation appears to be a characteristic of ETTs, even in the absence of LPCAT1-TERT fusions, and that it appears linked to copy number gains of chromosome 5. No evidence of TERT upregulation was identified in other trophoblastic lesions tested, including placental site trophoblastic tumors and placental site nodules, which are thought to be the benign chorionic-type trophoblast counterpart to ETT. These findings indicate that LPCAT1-TERT fusions and copy-number driven TERT activation may represent novel markers for ETT, with the potential to improve the diagnosis, treatment, and outcome for women with this rare form of GTD.

6072 Background: Effective screening tests for ovarian cancer (OC) are lacking; most cases present at advanced stage and portend poor prognosis. DNA methylation is an early event in carcinogenesis and can be detected in blood plasma samples from cancer patients. In DNA extracted from tissues, we first discovered, then validated discriminant methylated DNA marker (MDM) candidates for OC and subsequently tested independent plasma from women with and without OC. Methods: For discovery, DNA from 67 frozen tissues (18 high grade serous (HGS), 18 endometrioid, 15 clear cell (CC), 6 mucinous OCs; 10 benign fallopian tube epithelium (FT); and 19 buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify MDMs associated with OC. Candidate MDM selection was based on receiver operating characteristic (ROC) discrimination, methylation fold change, and low background methylation among controls. Blinded biological validation was performed using methylated specific PCR on DNA extracted from independent FFPE tissues from OCs (36 HGS, 22 endometrioid, 21 CC, and 14 mucinous) and 29 FT. Top performing MDMs in tissue were tested using long-probe quantitative amplified signal assays in independent pre-treatment plasma samples from women newly-diagnosed with OC and population-sampled healthy women. A random forest modeling analysis was performed to generate predictive probability of disease; results were 500-fold in silico cross-validated. Results: After RRBS discovery and biological validation, 33 MDMs showed marked methylation fold changes (10 to > 1000) across all OC histologies vs FT. The top 11 MDMs ( GPRIN1, CDO1, SRC, SIM2, AGRN, FAIM2, CELF2, DSCR6, GYPC, CAPN2, BCAT1) were tested on plasma from 91 women with OC (76 (84%) HGS) and 91 without OC; the cross-validated 11-MDM panel highly discriminated OC from controls (96% (95%CI 89-99%) specificity; 79% (69-87%) sensitivity, and AUC 0.91 (0.86 - 0.96)). Among HGS, the panel correctly identified 83%, including 5/6 stage I/II, and the majority of other subtypes (Table). Conclusions: Whole methylome sequencing, stringent filtering criteria, and biological validation yielded outstanding candidate MDMs for OC that performed with promisingly high sensitivity and specificity in plasma. Larger plasma-based OC MDM testing studies, with larger numbers of non-HGS histologies are warranted. [Table: see text]

Key Points Maternal T cells drive VUE responses; fetal immune cells drive infectious etiology. Clonal overlap between VUE subjects is minimal but common in placental infection. Contrary to placental infection, VUE T cells do not recognize common viral epitopes. During human pregnancy, proinflammatory responses in the placenta can cause severe fetal complications, including growth restriction, preterm birth, and stillbirth. Villitis of unknown etiology (VUE), an inflammatory condition characterized by the infiltration of maternal CD8+ T cells into the placenta, is hypothesized to be secondary to either a tissue rejection response to the haploidentical fetus or from an undiagnosed infection. In this study, we characterized the global TCR β-chain profile in human T cells isolated from placentae diagnosed with VUE compared with control and infectious villitis–placentae by immunoSEQ. Immunosequencing demonstrated that VUE is driven predominantly by maternal T cell infiltration, which is significantly different from controls and infectious cases; however, these T cell clones show very little overlap between subjects. Mapping TCR clones to common viral epitopes (CMV, EBV, and influenza A) demonstrated that Ag specificity in VUE was equal to controls and significantly lower than CMV-specific clones in infectious villitis. Our data indicate VUE represents an allograft response, not an undetected infection. These observations support the development of screening methods to predict those at risk for VUE and the use of specific immunomodulatory therapies during gestation to improve outcomes in affected fetuses.

Purpose: molecular testing is often indicated for recently transfused patients. However, there are no guidelines regarding the potential interference from donor DNA or whether it is necessary to wait for a period of time post-transfusion prior to genetic testing. While the majority of patients are transfused in the non-trauma setting using leukoreduced (LR) red blood cell products, the degree of leukoreduction varies among centers and is not universally practiced. Methods: whole blood units collected from anonymous donors were used in an in vitro transfusion model. One unit was split: half being leukoreduced simulating a leukopenic recipient and half left untreated. Donors were simulated by leukoreduced, partially leukoreduced (PLR), or non-leukoreduced units, transfused in 2, 5, or 16 unit equivalents. DNA from the combinations were subjected to short tandem repeat (STR) analysis for chimerism detection. Results: donor DNA was not detectable in any of the LR combinations, but detected in the PLR combinations, ranging from 0.1 to 1.5% donor DNA in the immunocompetent recipient and 6.3–27.8% in the leukopenic recipient. Non-LR donor DNA was also detected (13–95%). Conclusion: donor-derived DNA from leukoreduced blood products is unlikely to interfere with the interpretation of germline genetic testing in immunocompetent recipients but may interfere in immunocompromised recipients.

Despite the current classification of high-grade serous carcinoma (HGSCA) and low-grade serous carcinoma (LGSCA) as mutually exclusive diseases based on morphology and molecular pathogenesis, cases with mixed morphologic features of HGSCA and LGSCA have been reported. Herein we assess the clinicopathologic, immunohistochemical (IHC), and molecular genetic characteristics of a group of these cases, which we termed indeterminate grade serous carcinoma (IGSCA) in comparison with groups of HGSCA and LGSCA. Using the World Health Organization (WHO) classification criteria, we selected 27 LGSCA and 19 IGSCA for detailed morphologic study. Thirteen classic HGSCA, 19 classic LGSCA, and 19 IGSCA were selected for p53 and BRAF V600E IHC and molecular genetic testing by next-generation sequencing. IGSCA showed the architectural patterns of invasion of LGSCA, but with higher grade nuclear features focally and a mitotic index intermediate between LGSCA and HGSCA. Few cases in the IGSCA group showed mutant TP53 by IHC or sequencing (4/18, 22.2%), 1 case had mutant BRAF non-V600E by sequencing, and 1 had an NRAS mutation. When present, the mutations were identical in the low-grade and high-grade areas. The IGSCA group had a long-term survival similar to the classic HGSCA group. IGSCA with mixed morphologic features of HGSCA and LGSCA is a rare and potentially clinically aggressive variant of serous carcinoma. Their distinct morphologic, but heterogenous molecular features, including low frequency of TP53 and BRAF mutations suggest that these rare tumors may have a different pathogenesis pathway compared with classic HGSCA and classic LGSCA.