Navigating Practice Issues Related to the Unsatisfactory Cervicovaginal Papanicolaou Test: Survey Results of Laboratories Participating in the 2020 College of American Pathologists PAP Education Program.

CONTEXT.­: Unsatisfactory Papanicolaou (Pap) tests pose a unique set of challenges to the laboratory with regard to their processing, review, reporting, and performance of human papillomavirus (HPV) testing. There are no standardized guidelines for the review process and handling of unsatisfactory Pap tests. OBJECTIVE.­: To assess the current practice patterns regarding various aspects of the unsatisfactory Pap test, from processing to reporting, across laboratories worldwide. DESIGN.­: A supplemental questionnaire was mailed to laboratories participating in the 2020 College of American Pathologists (CAP) Gynecologic Cytopathology (PAP Education) Program, requesting data regarding the unsatisfactory Pap test. RESULTS.­: Of 1520 participating laboratories, 619 (40.7%) responded, and the responses of 577 laboratories were included for further analysis. Only 64.6% (373 of 577) laboratories used the unsatisfactory Pap test criteria as specified by the 2014 Bethesda System. About three-quarters of the respondents (433 of 576; 75.2%) routinely rescreened unsatisfactory Pap tests. Routine repreparation of such Pap tests was performed by 54.9% (316 of 576) of laboratories, and 52.0% (293 of 563) used glacial acetic acid for repreparing excessively bloody specimens. HPV test results were reported for unsatisfactory Pap tests, always or sometimes, by 62.4% (353 of 566) of respondents. CONCLUSIONS.­: This CAP survey reveals important information regarding the practice patterns pertaining to several aspects of the unsatisfactory Pap test. It also provides valuable insight into the quality assurance measures that can be implemented for such tests. Future studies can further aid in the standardization of all components of the handling of unsatisfactory Pap tests for overall quality improvement.

Women 50 Years and Older With Negative Pap Test and Positive Human Papillomavirus Test for Genotypes Other Than 16 and 18-Follow-up Outcomes.

OBJECTIVE: A follow-up of women 50 years or older with concomitant positive high-risk human papillomavirus (HPV) genotypes other than 16 and 18 (hrHPVO) and negative Pap test (NILMPap) was conducted to better understand the implications of hrHPVO positivity on potential risk of developing significant high-grade lesions. MATERIAL AND METHODS: A retrospective review of 2014 cytology data of patients with co-testing (Pap test and HPV DNA) identified 85 women 50 years or older with NILMPap and hrHPVO+. RESULTS: Most patients (63) had repeat co-testing on next follow-up. Of these, 41 patients with persistent hrHPVO+ status, 3 developed cervical intraepithelial neoplasia 2 (CIN2), and 1 CIN3. Nineteen patients were followed with biopsies. Of these, 7 biopsies were abnormal, 5 of which showed low-grade (CIN1) and 2 high-grade (CIN3) histology; none progressed on further follow-up. Three patients were followed with Pap test only, all had NILMPap, and none progressed on further follow-up. In summary, of the 85 patients, 26 developed abnormal histology during follow-up, 6 of whom had high-grade histology (CIN2 and CIN3, 3 each).The 5-year risk of CIN1+ in this cohort was 43.8% and for CIN2+ was 12.3%. The risk of abnormal histology did not differ significantly by prior history of Pap tests, histology, and/or HPV results. CONCLUSIONS: A persistent positivity for hrHPVO indicated higher likelihood to develop a lesion, and this risk was not reduced for patients 50 and older compared with the published screening population risk.

Tao brush endometrial cytology is a sensitive diagnostic tool for cancer and hyperplasia among women presenting to clinic with abnormal uterine bleeding.

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.

Evaluation of high-risk human papillomavirus testing and anal cytology to detect high-grade anal intraepithelial neoplasia.

INTRODUCTION: Optimal screening for detection of anal precancer has not been established, and most studies involve very high-risk populations. We evaluated high-risk human papillomavirus (HPV) testing and anal cytology to detect high-grade anal intraepithelial neoplasia (≥AIN2) in a cohort with mostly moderate risk factors for AIN. METHODS: Patients ≥35 years old undergoing anal biopsy for various lesions received HPV testing by Roche cobas and a subset by Hologic APTIMA HPV assays with concurrent anal ThinPrep cytology. Biopsies were blindly reviewed by 3 authors, and consensus diagnosis was compared with HPV and cytology results. Sensitivity and specificity for ≥AIN2 detection by HPV testing and cytology (≥ASC-US) were calculated. RESULTS: Among 64 patients, 19 (29.7%) showed ≥AIN2 on biopsy. All patients were tested by cobas, and 35 (54.7%) were positive. A subset of 39 patients were also tested by APTIMA, and 18 (46.2%) were positive. Positive cytology (≥ASC-US) was present in 37 (57.8%) patients, with 27 (73.0%) of these positive by cobas. HPV testing alone yielded 75.0% and 84.2% sensitivity for APTIMA and cobas, respectively; specificity was 66.7% and 57.8%. Sensitivity and specificity of cytology alone was 78.9% and 51.1%. Combined HPV testing and cytology had a sensitivity and specificity of 91.7% and 37.0% for APTIMA and 94.7% and 40.0% for cobas. CONCLUSIONS: Combined HPV testing and cytology had the highest sensitivity for ≥AIN2 detection, with a performance comparable to cervical cancer screening tests, suggesting this strategy may represent a viable screening option in a population with moderate risk factors for AIN.

Development of a technique for evaluating the presence of malignant cells in prostatic fluid during robotic prostatectomy.

INTRODUCTION: To develop a technique to collect fluid expressed during robot-assisted radical prostatectomy (RARP) to assess whether malignant cells may have been inadvertently introduced into the surgical field. METHODS: Men with clinically localized grade group 2 to 5 prostate adenocarcinoma undergoing RARP were identified. Following bladder neck division, fluid expressed via prostatic urethra during seminal vesicle dissection was aspirated (specimen A). After specimen removal, an ex vivo seminal vesicle aspiration was performed as well (specimen B). Specimens were prepared with ThinPrep (Hologic, Marlborough, MA) and stained with 4 immunohistochemical markers: keratin-7, PAX-8, prostate-specific antigen (PSA), and prostatic acid phosphatase (PACP). RESULTS: Between December 2018 and May 2019, 15 men undergoing RARP were included. Median age was 60 years (range: 47-77), median PSA 8.5 ng/ml (range 5.1-24), and 7 (47%) had AUA high-risk disease. Specimen A had adequate cellularity in 13 patients (87%). Five patients were excluded from assessment of malignancy due to acellularity of specimen A (n = 2) or specimen B (n = 3). Three of the remaining 10 patients (30%) had cytologic features suspicious for malignancy on specimen A. Immunohistochemistry supported prostatic origin with positive PSA and PACP staining and negative PAX8 stains. Specimen B was not suspicious in any patient. CONCLUSION: We report a technique for intraoperative collection of fluid expressed during RARP. Three patients with adverse pathologic features had evidence of cancer cells within the operative field. Further work is needed to confirm this observation and to determine whether these cells are associated with adverse oncologic outcomes.

Retrospective 15-Year Review of Anal Cytology Screening in Women at Mayo Clinic Rochester, Minnesota.

OBJECTIVES: Anal cytology is a modality for anal cancer screening in high-risk women. In this retrospective study, we review risk factors associated with abnormal anal cytology and unsatisfactory anal cytology rates, and correlate findings of cytology with histological results. METHODS: A retrospective cohort study of anal cytology screening in women at Mayo Clinic in Rochester, Minnesota from 2002 to 2018 was conducted. RESULTS: Three hundred fifty-seven women had a total of 592 anal cytologies performed. Three hundred seventeen women had screening anal cytology, whereas 40 women had anal cytology for surveillance given a history of anal intraepithelial neoplasia (AIN) or anal cancer. An unsatisfactory anal cytology result was found in 14.7%. Risk factors, type of follow-up, and correlation with histologic specimens were also reviewed. Histologic finding of AIN 2/3 correlated with abnormal anal cytology 84% of the time in this cohort. CONCLUSIONS: High-risk women should be screened on a periodic basis for anal cancer. Anal cytology is one possible modality that can be used. Further insight into AIN progression, regression, recurrence, and outcome after treatment will help direct future screening recommendations.

Clinical impact of celiac ganglia metastasis upon pancreatic ductal adenocarcinoma.

BACKGROUND: Pre-operative staging of pancreatic adenocarcinoma guides clinical decision making. Limited data indicate that metastasis to celiac ganglia (CG) correlates with poor prognosis. We investigated feasibility and safety of endoscopic ultrasound fine needle aspiration (EUS-FNA) detection of CG metastasis and its impact upon tumor stage, resectability, and survival in pancreatic ductal adenocarcinoma (PDAC). PATIENTS: We reviewed our prospectively maintained EUS and cytopathology databases to identify patients with FNA proven CG metastasis in patients with PDAC from 2004 to 2017. Clinical demographics, EUS, CT, MRI, cytopathology, cancer stage, and resectability data were analyzed. Survival of PDAC patients with CG metastasis was compared to the expected survival of PDAC patients of similar stage as reported by the United States National Cancer Database. RESULTS: Twenty-one patients with PDAC [median age 73 (IQR63-78); 14 (67%) female)], had CG metastasis confirmed by cytopathologic assessment. CG metastasis resulted in tumor upstaging relative to other EUS findings and cross sectional imaging findings in 12 (57%) and 15 (71%) patients, and converted cancers from resectable to unresectable relative to EUS and cross sectional imaging in 7 (37%) and 7 (37%) patients, respectively. In patients with PDAC, the survival of patients with CG metastasis was not significantly different from the overall survival (hazard ratio 0.71; 95% confidence interval 0.44, 1.13; p = 0.15). CONCLUSIONS: EUS-FNA may safely identify CG metastases. While CG metastasis upstaged and altered the resectability status among this cohort of patients with PDAC, the survival data with regard to PDAC suggest that this may be misguided.

Assessment of The Bethesda System for Reporting Thyroid Cytopathology.

OBJECTIVES: Long-term follow-up is important for determining performance characteristics of thyroid fine-needle aspiration (FNA). METHODS: Histologic or 3 or more years of clinical follow-up was used to calculate performance characteristics of thyroid FNA before and after implementation of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC). The impact of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) classification was also investigated. RESULTS: Follow-up was obtained for 1,277/1,134 and 1,616/1,393 aspirates/patients (median clinical follow-up, 9.9 and 4.4 years, pre- and post-TBSRTC, respectively). Nondiagnostic, suspicious for follicular neoplasm, and suspicious for malignancy (SFM) diagnoses decreased and benign diagnoses increased post-TBSRTC, while atypical rate remained less than 1%. Negative predictive value for benign nodules and positive predictive value (PPV) for SFM increased significantly. Eleven nodules were reclassified as NIFTP, slightly decreasing PPV/risk of malignancy (ROM). CONCLUSIONS: Appropriate ROM for thyroid FNA can be achieved through application of TBSRTC terminology with minimal use of atypical category.

Diagnostic value of imprint cytology testing in kidney tumors: review of 200 cases.

INTRODUCTION: Previous investigations have studied the importance of imprint cytology (IC) testing of core needle biopsy (CNB) from various organs. We have presented the largest series, to the best of our knowledge, of IC testing of CNB for patients with kidney tumors. MATERIALS AND METHODS: The present retrospective study (January 1, 2015, through January 30, 2016) identified laboratory information through a computer search of the cytology archived reports for 200 consecutive IC testing with CNB for renal tumors cases. A board-certified cytopathologist and cytology-trained fellow reviewed the IC testing and CNB slides and rendered them as nondiagnostic, positive for malignancy, negative for malignancy, positive for neoplasm, or atypical. The tumors were graded using the International Society of Urological Pathology grading system. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated. RESULTS: The IC testing cases classified as atypical (n = 53) or positive for neoplasm (n = 28) were evaluated separately because of the ambiguous morphologic characteristics. Of the other 119 cases, IC testing classified 95 (80%) as positive for malignancy, 5 (4%) as negative for malignancy, and 19 (16%) as nondiagnostic. The corresponding CNB histologic diagnoses showed that 85 of 95 cases (89%) were true positive for malignancy. Of these 85 cases, 45 (53%) were low grade, 21 (25%) were high grade, and 19 (22%) were ungraded. The corresponding sensitivity, specificity, and accuracy were 85%, 11%, and 58%, respectively. The 53 IC-identified atypical cases were more likely to be malignant (n = 40; 75%). Of the remaining IC testing atypical cases, 12 (23%) were negative for malignancy and 1 (2%) was nondiagnostic. Of the 28 cases positive for neoplasm using IC, 13 (46%) were positive and 15 (54%) were negative for malignancy. CONCLUSIONS: The relatively low diagnostic value of IC testing for renal tumors showed it to be less powerful for screening than its use in other organs.

Accuracy of Endoscopic Ultrasound Imaging in Distinguishing Celiac Ganglia From Celiac Lymph Nodes.

BACKGROUND & AIMS: Endoscopic ultrasound (EUS) allows visualization of celiac lymph nodes (CLNs) and celiac ganglia (CG). Reliably distinguishing these structures is important for tumor staging and CG ablative therapies. We aimed to evaluate the accuracy of EUS in distinguishing CLNs from CG using a strict cytopathology reference standard. We also determined the rate of detection of CLN and CG by conventional cross-sectional imaging. METHODS: From EUS and cytopathology databases, we identified all patients who underwent EUS-FNA of a presumed CLN or CG from October 1, 2004, through March 1, 2017, and compared the findings with those from cytology (reference standard). Indeterminate cytology results were re-reviewed. EUS imaging (ie, index test) results were compared with those from the reference standard. An expert radiologist re-reviewed computed tomography and magnetic resonance images from 100 lesions, from 94 randomly selected patients with a reference standard, to determine the rates of CLN and CG detection. RESULTS: A total of 504 patients (mean age, 63.4 ± 13.2 years; 292 men) underwent a median of 7 EUS-FNA passes (range, 1-13) for a total of 566 lesions perceived to be either a CLN or CG; the cytology reference standard was available for 521 lesions (92.1%). When we excluded indeterminate cytology results, the EUS accurately identified 281/286 CLNs (98.3%) and 166/186 CGs (89.2%), for an overall accuracy of 447/472 (94.7%). EUS-FNA distinguished CG from CLNs with a 93.3% sensitivity, 93.7% specificity, a positive predictive value of 96.2%, and a negative predictive value of 89.2%. Of 100 lesions in 94 patients randomly selected for a second expert radiology review, computed tomography and magnetic resonance imaging detected 59/67 CLNs (88.1%) and 13/33 CG (39.4%). CONCLUSION: EUS accurately distinguishes CLNs from CG. EUS might therefore be used to increase the accuracy of tumor staging, to select tumor stage-appropriate therapy, and to guide CG-ablative therapies.

Analysis of Cell-Free DNA to Assess Risk of Tumoremia Following Endoscopic Ultrasound Fine-Needle Aspiration of Pancreatic Adenocarcinomas.

BACKGROUND & AIMS: Cellular and nuclear material from tumors disseminates into the bloodstream (tumoremia), but it is not clear whether medical procedures cause release of this material or contribute to formation of metastases. We performed a prospective study of blood samples from patients with pancreatic adenocarcinoma (PDAC) to determine whether endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) associates with markers of tumoremia. METHODS: We obtained peripheral blood from 104 patients (35 with PDAC) before and after EUS-FNA of primary tumors; blood samples from 69 healthy individuals were used as controls. Plasma concentrations of cell-free DNA (cfDNA) were measured, and cfDNA and primary tumor samples were analyzed to detect activating mutations in KRAS. Potential development of tumoremia was defined by an increase in cfDNA of 2-fold or more, and/or detection of mutant KRAS in samples collected after FNA from patients whose blood samples did not contain detectable mutant KRAS before FNA. RESULTS: Peripheral blood concentrations of cfDNA were 1200 ng/ml (500-3300 ng/ml) before FNA vs 1400 ng/ml (900-4000 ng/ml) after FNA (P = .391). Tumoremia was detected in 10/35 patients (28.6%): 7 patients had a ≥2-fold increase in cfDNA concentration (20.6%) and 3 patients had circulating tumor DNA with KRAS mutations after FNA that were not detected before FNA (8.8%). New distant metastases were detected in 1.3 ± 0.82 patients with tumoremia vs 0.64 ± 0.81 without (P = .0375). Overall mortality did not differ significantly between patients with tumoremia (10/10 deaths, 100%) vs those without (19/25 deaths, 76%) nor did survival times of deceased patients (13.3 months for patients with tumoremia; range, 5.8-14.9 months vs 11.1 months for patients without tumoremia; range, 5.5-14.5 months). However, 6 patients without tumoremia were alive at a mean 23.9 months after EUS-FNA (range, 19.9-25 months after EUS-FNA) vs none of the patients with tumoremia. CONCLUSION: In patients with PDAC, EUS-FNA associates with increased plasma concentration of cfDNA and increased detection of mutant KRAS after the procedure (markers of tumoremia and possible new distant metastasis). Although levels of cfDNA and activating mutations in KRAS are logical markers of tumoremia, they may not serve as the ideal biomarkers of this process. These findings are preliminary and do not indicate a need to modify current practice, yet further studies are needed.

High-risk HPV genotype distribution in HPV co-test specimens: study of a predominantly Midwestern population.

INTRODUCTION: High-risk HPV (HR-HPV) genoprevalence was assessed in cytologic specimens co-tested for HR-HPV in a predominantly Midwestern U.S. population, of which there are limited current data. These baseline data will aid in determining future shifts in HR-HPV genoprevalence. METHODS: A total of 40,739 cervical/endocervical samples collected in PreservCyt (ThinPrep) media at Mayo Clinic, Rochester, MN, underwent HR-HPV co-testing between January 2014 and December 2016 in women aged 30-98 years. This included 37,656 negative for intraepithelial lesion (NIL), 1696 atypical squamous cells of undetermined significance (ASC-US), 159 atypical squamous cells, cannot exclude HSIL (ASC-H), 911 low-grade squamous intraepithelial lesion (LSIL), 188 high-grade intraepithelial lesion (HSIL), and 129 atypical glandular cells of undetermined significance (AGUS) cases. Roche cobas 4800 classified HR-HPV genotypes as 16, 18, or "other" (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68). RESULTS: Of 40,739 co-tested specimens, 3786 were positive for at least one HR-HPV strain. "Other" only genotypes were most prevalent (74.3% of all HR-HPV cases). HPV16 and/or HPV18 were more common in the more significant diagnoses and were present in 59.1% of HSIL. HPV16-only was second most prevalent, with highest prevalence in HSIL (33.3%) and ASC-H (20.6%). HPV16 combined with "other" was third most prevalent, except in AGUS. HR-HPV (all genotypes) was most prevalent in those aged 30-39 years, decreasing with age (P < 0.0001). There was a trend toward HPV16 prevalence increasing with age (P = 0.4244). CONCLUSIONS: HR-HPV "other" combination was most prevalent in all diagnoses except HSIL, in which HPV16 and/or HPV18 (exclusive of "other" genotypes) were most prevalent.

Assessment of pancreatic neuroendocrine tumor cytologic genotype diversity to guide personalized medicine using a custom gastroenteropancreatic next-generation sequencing panel.

BACKGROUND: Recent genetic studies have highlighted that alterations in MEN1, chromatin remodeling genes, and mammalian target of rapamycin (mTOR) pathway genes are the most frequent molecular events identified in pancreas neuroendocrine tumors (pNETs). The prognostic or predictive impact of these biomarkers and other less frequently observed aberrations, i.e. PTEN, TSC2 and PIK3CA are relatively unknown. The aims of this targeted next generation sequencing (NGS) study were to assess tumor cytology genotype diversity, to survey for potential adverse prognostic biomarkers and the prevalence of mTOR pathway variants. METHODS: Using a custom 15 gene gastroenteropancreatic neuroendocrine tumor panel, targeted NGS of archived (2002-2013) primary pNETs (n=90) and pNET liver metastasis (n=32) cytology smears was performed. RESULTS: The genetic variant landscape revealed that 21% and 28% of primary and metastatic liver pNETs harbored ≥ 2 variants per tumor, respectively. The most prevalent primary tumor variants were in the MEN1 (42%), DAXX (11%), ATRX (10%), and TSC2 (8%) genes. Patients harboring aberrations in TSC2, KRAS or TP53 were more likely to experience disease progression and reduced overall survival, when compared to individuals who were wild-type. The prevalence of these potential prognostic biomarkers in early disease was observed in 3.3% of the primary tumor cohort. mTOR pathway variants including alterations in PTEN, TSC2 and PIK3CA were identified in 10% and 12.5% of primary tumors and pNET liver metastasis, respectively. CONCLUSION: Cytology based tumor genotyping revealed a broad spectrum of genetic variants including possible adverse prognostic biomarkers, reflective of an aggressive phenotype. It also demonstrated the prevalence of potential predictive biomarkers for mTOR pathway inhibitor sensitivity.

Oral Progestogens Versus Levonorgestrel-Releasing Intrauterine System for Treatment of Endometrial Intraepithelial Neoplasia.

BACKGROUND: Limited therapeutic guidelines exist regarding medical therapy, ideal dosing, duration of therapy, or recommendations for timing of endometrial reassessment for women with endometrial intraepithelial neoplasia (EIN) who desire fertility preservation or who are not optimal surgical candidates. We aimed to determine the effectiveness of oral progestogens (OP) versus the levonorgestrel-releasing intrauterine system (LNG IUS) in the medical treatment of EIN. METHODS: We retrospectively identified women with EIN at our institution from 2007 through 2014 and compared the outcomes of those treated with OP versus LNG IUS. RESULTS: Among 390 women, 296 were initially treated with OP and 94 with LNG IUS. Baseline characteristics of the patient groups were comparable, except for higher median body mass index in the LNG IUS group versus the OP group (37 kg/m2 vs. 31 kg/m2; p < 0.001). Among 332 women with follow-up endometrial biopsies (263 OP and 69 LNG IUS), EIN subcategory 1 (benign endometrial hyperplasia) resolved in 83% and 87% of patients, respectively (p = 0.31). Rates of resolution of EIN subcategory 2 (endometrial intraepithelial neoplasia) were also similar between groups (68% vs. 62%; p = 0.82). In women with EIN subcategory 3 (endometrial adenocarcinoma), 22% of those using LNG IUS and one of two women treated with OP had resolution of disease as of last follow-up. CONCLUSIONS: OP and LNG IUS offer similar endometrial protection for women with EIN. LNG IUS offers convenience, minimal adverse effects, reversibility, and long-term endometrial protection.

Targeted next generation sequencing of endoscopic ultrasound acquired cytology from ampullary and pancreatic adenocarcinoma has the potential to aid patient stratification for optimal therapy selection.

BACKGROUND & AIMS: Less than 10% of registered drug intervention trials for pancreatic ductal adenocarcinoma (PDAC) include a biomarker stratification strategy. The ability to identify distinct mutation subsets via endoscopic ultrasound fine needle aspiration (EUS FNA) molecular cytology could greatly aid clinical trial patient stratification and offer predictive markers. We identified chemotherapy treatment naïve ampullary adenocarcinoma and PDAC patients who underwent EUS FNA to assess multigene mutational frequency and diversity with a surgical resection concordance assessment, where available. METHODS: Following strict cytology smear screening criteria, targeted next generation sequencing (NGS) using a 160 cancer gene panel was performed. RESULTS: Complete sequencing was achieved in 29 patients, whereby 83 pathogenic alterations were identified in 21 genes. Cytology genotyping revealed that the majority of mutations were identified in KRAS (93%), TP53 (72%), SMAD4 (31%), and GNAS (10%). There was 100% concordance for the following pathogenic alterations: KRAS, TP53, SMAD4, KMT2D, NOTCH2, MSH2, RB1, SMARCA4, PPP2R1A, PIK3R1, SCL7A8, ATM, and FANCD2. Absolute multigene mutational concordance was 83%. Incremental cytology smear mutations in GRIN2A, GATA3 and KDM6A were identified despite re-examination of raw sequence reads in the corresponding resection specimens. CONCLUSIONS: EUS FNA cytology genotyping using a 160 cancer gene NGS panel revealed a broad spectrum of pathogenic alterations. The fidelity of cytology genotyping to that of paired surgical resection specimens suggests that EUS FNA represents a suitable surrogate and may complement the conventional stratification criteria in decision making for therapies and may guide future biomarker driven therapeutic development.

Molecular cytology genotyping of primary and metastatic GI stromal tumors by using a custom two-gene targeted next-generation sequencing panel with therapeutic intent.

BACKGROUND AND AIMS: In an era of precision medicine, customized genotyping of GI stromal tumors by screening for driver mutations will become the standard of care. The fidelity of genotype concordance between paired cytology smears and surgical pathology specimens is unknown. In patients with either primary or metastatic sporadic disease, we sought to determine the frequency of KIT and PDGFRA pathogenic alterations within such specimens, imatinib sensitivity, and the concordance of pathogenic alterations between paired specimens. METHODS: DNA obtained from cytology smears from 36 patients, 24 of whom had paired surgical pathology specimens, underwent targeted next-generation sequencing by using a custom panel to evaluate somatic mutations within KIT (exon 2, 9, 10, 11, 13, 14, 15, 17, 18) and PDGFRA (exon 12, 14, 15, 18) genes. Patients with KIT and PDGRFA wild-type genes completed the Qiagen Human Comprehensive Cancer GeneRead DNAseq Targeted Array V2. RESULTS: Genotyping revealed KIT and PDGFRA mutations in 68% and 15% of patients. The wild-type population did not harbor mutations in BRAF, RAS family, SDHB, SETD2, or NF1. Imatinib sensitivity based on the oncogenic kinase mutation prevalence was estimated to be 68%. Mutational concordance between paired cytology and surgical pathology specimens was 96%. CONCLUSIONS: Our data have demonstrated the ability to stratify either primary or metastatic gastrointestinal stromal tumors by mutational subtype using a targeted next-generation sequencing 2 gene mutation panel. We highlight the ability to use cytology specimens obtained via minimally invasive techniques as a surrogate to surgical specimens given the high mutational landscape concordance between paired specimens.

Young investigator challenge: Validation and optimization of immunohistochemistry protocols for use on cellient cell block specimens.

BACKGROUND: The objective of the current study was to establish a process for validating immunohistochemistry (IHC) protocols for use on the Cellient cell block (CCB) system. METHODS: Thirty antibodies were initially tested on CCBs using IHC protocols previously validated on formalin-fixed, paraffin-embedded tissue (FFPE). Cytology samples were split to generate thrombin cell blocks (TCB) and CCBs. IHC was performed in parallel. Antibody immunoreactivity was scored, and concordance or discordance in immunoreactivity between the TCBs and CCBs for each sample was determined. Criteria for validation of an antibody were defined as concordant staining in expected positive and negative cells, in at least 5 samples each, and concordance in at least 90% of the samples total. Antibodies that failed initial validation were retested after alterations in IHC conditions. RESULTS: Thirteen of the 30 antibodies (43%) did not meet initial validation criteria. Of those, 8 antibodies (calretinin, clusters of differentiation [CD] 3, CD20, CDX2, cytokeratin 20, estrogen receptor, MOC-31, and p16) were optimized for CCBs and subsequently validated. Despite several alterations in conditions, 3 antibodies (Ber-EP4, D2-40, and paired box gene 8 [PAX8]) were not successfully validated. CONCLUSIONS: Nearly one-half of the antibodies tested in the current study failed initial validation using IHC conditions that were established in the study laboratory for FFPE material. Although some antibodies subsequently met validation criteria after optimization of conditions, a few continued to demonstrate inadequate immunoreactivity. These findings emphasize the importance of validating IHC protocols for methanol-fixed tissue before clinical use and suggest that optimization for alcohol fixation may be needed to obtain adequate immunoreactivity on CCBs.