Ninjurin 1 has two opposing functions in tumorigenesis in a p53-dependent manner.

WT p53 is critical for tumor suppression, whereas mutant p53 promotes tumor progression. Nerve injury-induced protein 1 (Ninj1) is a target of p53 and forms a feedback loop with p53 by repressing p53 mRNA translation. Here, we show that loss of Ninj1 increased mutant p53 expression and, subsequently, enhanced cell growth and migration in cells carrying a mutant p53. In contrast, loss of Ninj1 inhibited cell growth and migration in cells carrying a WT p53. To explore the biological significance of Ninj1, we generated a cohort of Ninj1-deficient mice and found that Ninj1+/- mice were prone to systemic inflammation and insulitis, but not to spontaneous tumors. We also found that loss of Ninj1 altered the tumor susceptibility in both mutant p53 and p53-null background. Specifically, in a mutant p53(R270H) background, Ninj1 deficiency shortened the lifespan, altered the tumor spectrum, and increased tumor burden, likely via enhanced expression of mutant p53. In a p53-null background, Ninj1 deficiency significantly increased the incidence of T-lymphoblastic lymphoma. Taken together, our data suggest that depending on p53 genetic status, Ninj1 has two opposing functions in tumorigenesis and that the Ninj1-p53 loop may be targeted to manage inflammatory diseases and cancer.

HPV 6-associated HSIL/Squamous Carcinoma in the Anogenital Tract.

Human papillomavirus (HPV) type 6 is historically classified as low-risk HPV type and associates with low-grade squamous intraepithelial lesions of the anogenital tract. Rare squamous carcinomas have been reported in association with these HPV types but the mechanism(s) behind this carcinogenic sequence have been unclear. We report 4 cases of low risk anogenital HPV infections-3 cervical (immature low-grade squamous intraepithelial lesion with metaplastic phenotype) and one anal (exophytic condyloma) lesion-that manifested with high-grade squamous intraepithelial lesion/squamous cell carcinoma. Two were associated with invasion one of which metastasized to a regional node. Two cases exhibited strong p53 positivity in the high-grade squamous intraepithelial lesion/squamous cell carcinoma component analogous to that seen in HPV-negative differentiated intraepithelial lesions of the external genitalia. This series of cases adds to the literature on low risk HPV-associated cervical squamous carcinomas. It underscores the similarities between the baseline cyto-morphology and benign mimics (low-grade squamous intraepithelial lesions), the subtle cytologic and immunohistochemical (MIB1) features heralding biologic aggressiveness, and in some potential pathways (p53) not usually involved in HPV-related anogenital neoplasia.

Novel heterozygous mutation in the PTEN gene associated with ovarian germ cell tumor complicated by growing teratoma syndrome and overgrowth in a two-year-old female.

Growing teratoma syndrome (GTS) is a condition in which mature teratoma with negative tumor markers arises at the site of a treated malignant germ cell tumor. Pathogenic variants in PTEN have been reported to cause autosomal dominant cancer predisposition syndromes and are associated with germ cell tumors. We report the association of a novel heterozygous pathogenic variant in PTEN and very early onset ovarian germ cell tumor complicated by GTS as well as overgrowth syndrome. This marks the youngest reported patient to have developed GTS following treatment of her primary malignant ovarian germ cell tumor.

Patients with Non-invasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features are Unlikely to have Malignant Preoperative Cytology.

BACKGROUND: The newly termed tumor 'noninvasive follicular thyroid neoplasm with papillary-like nuclear features' (NIFTP) acts in an indolent manner and can likely be safely managed with a thyroid lobectomy. Preoperative fine-needle aspiration (FNA) is the cornerstone of surgical planning, but the ability of FNA to distinguish NIFTP from other variants of papillary thyroid carcinoma (PTC) has not been well-evaluated. METHODS: A 9-year retrospective review of the preoperative cytology and surgical pathology of PTC patients who underwent a thyroidectomy at our tertiary referral center. RESULTS: Overall, 174 patients with 177 PTCs had a preoperative FNA followed by a thyroidectomy, and met our inclusion criteria. Of the 21 patients with NIFTP, the preoperative cytology was read as malignant in three (14%), suspicious for malignancy in three (14%), follicular neoplasm in ten (48%), atypia of undetermined significance in four (19%), and benign in one (5%) nodule. When comparing patients with NIFTP with other variants of PTC, patients with NIFTP were younger (p = 0.023) and less likely to have malignant cytology (p < 0.001). On multivariable regression modeling, malignant cytology was independently associated with a decreased risk of a final diagnosis of NIFTP (odds ratio 0.064, 95% confidence interval 0.018-0.233, p < 0.001). CONCLUSIONS: Patients with a final diagnosis of NIFTP are less likely to have preoperative FNA diagnosis of malignancy than those with final pathology of classical or other variants of PTC. Surgeons should take this into consideration when considering between a lobectomy and total thyroidectomy for patients with suspected PTC.

The Use of Endometrial Cancer Patient-Derived Organoid Culture for Drug Sensitivity Testing Is Feasible.

OBJECTIVE: Patient-derived organoids (PDOs), used in multiple tumor types, have allowed evaluation of tumor characteristics from individual patients. This study aimed to assess the feasibility of applying PDO in vitro culture for endocrine-based and drug sensitivity testing in endometrial cancer. METHODS: Endometrial cancer cells were enzymatically dissociated from tumors retrieved from fresh hysterectomy specimens and cultured within basement membrane extract in serum-free medium. An organoid growth assay was developed to assess the inhibitory effects of a variety of drugs including endocrine treatments. Organoid cultures were also prepared for histological and immunohistochemical comparison to the tumors of origin. RESULTS: Fifteen endometrial cancer specimens were successfully cultured as PDOs. Small spherical structures formed within 24 hours, and many continued to grow to larger, denser organoids, providing the basis for an organoid growth assay. The STAT3 transcription factor inhibitor, BBI608 (Napabucasin), strongly inhibited growth in almost all PDO cultures, suggesting that stemness programing is involved in organoid formation and/or growth. Inhibition by different growth factor receptor tyrosine kinase inhibitors was observed in several PDO specimens. Four cultures were inhibited by fulvestrant, implying the importance of estrogen-receptor signaling in some PDO cultures. Organoids closely resembled their tumors of origin in both histomorphology and immunohistochemical expression. CONCLUSIONS: The use of endometrial cancer PDO cultures for development of drug sensitivity testing for individual patient tumors is feasible. The potential value of the PDO model for clinical decision making will require clinical trial evaluation.

p16ink4 and cytokeratin 7 immunostaining in predicting HSIL outcome for low-grade squamous intraepithelial lesions: a case series, literature review and commentary.

p16ink4 and cytokeratin 7 (CK7) have been proposed to identify low-grade squamous intraepithelial lesions (LSIL) at greater or lesser risk for an outcome of high-grade squamous intraepithelial lesion (HSIL). We correlated CK7 and p16ink4 staining in LSILs with outcome on follow-up and placed this information in the context of prior reports. Cervical LSIL biopsies with at least 1-year follow-up information were immunostained for CK7 and p16ink4. Follow-up outcomes included no SIL, LSIL (persistence) or HSIL (CIN2+). In all, 109 LSILs were studied and 18.3% stained positive for CK7. Ninety-one percent of CK7-negative LSILs regressed, 4.5% persisted, and 4.5% had an HSIL outcome, versus 60, 20, and 20% of CK7-positive LSILs, respectively (P=0.036). p16ink4 status did not significantly associate with outcome. Review of the literature revealed a highly variable rate of both positive p16ink4 immunoreactivity in LSIL and CIN2+ outcome for p16-positive LSIL but a consistently high negative predictive value (>90%) in the case of no/low p16 expression. Inter-observer reproducibility for the diagnosis of CIN2 in the literature ranged from poor to good, with unanimous agreement on the diagnosis of CIN2 occurring in less than 25% of cases. As with high-risk human papillomavirus testing, the most clinically useful result of p16ink4 staining is a negative test, implying no lesion or CIN1 and conferring a low risk of HSIL outcome. HSIL outcomes ('progression') are highly variable and are subject to wide differences in inter-observer interpretation for CIN2. This argues against the wisdom of relying on p16ink4 to both predict CIN2+ or upgrade CIN1 to CIN2. It also begs the question of whether CIN2 should be replaced by an alternate and less pejorative term (SIL of intermediate grade) for lesions that are not reproducibly classified as LSIL or HSIL, with an appropriate management scheme.

Post-contrast myocardial T(1) and ECV disagree in a longitudinal canine study.

Both post-contrast myocardial T1 and extracellular volume (ECV) measurements have been associated with diffuse interstitial fibrosis. The cardiovascular magnetic resonance (CMR) field is migrating towards ECV, because it is largely insensitive to confounders that affect post-contrast myocardial T1 . Despite the theoretical advantages of myocardial ECV over post-contrast myocardial T1 , systematic experimental studies comparing the two measurements are largely lacking. We sought to measure the temporal changes in post-contrast myocardial T1 and ECV in an established canine model with chronic atrial fibrillation. Seventeen mongrel dogs, implanted with a pacemaker to induce chronic atrial fibrillation via rapid atrial pacing, were scanned multiple times for a total of 46 CMR scans at 3T. These dogs with different disease durations (0-22 months) were part of a separate longitudinal study aimed at studying the relationship between AF and pathophysiology. In each animal, we measured native and post-contrast T1 values and hematocrit. Temporal changes in post-contrast myocardial T1 and ECV, as well as other CMR parameters, were modeled with linear mixed effect models to account for repeated measurements over disease duration. In 17 animals, post-contrast myocardial T1 decreased significantly from 872 to 698 ms (p < 0.001), which corresponds to a 24.9% relative reduction. In contrast, ECV increased from 21.0 to 22.0% (p = 0.38), which corresponds to only a 4.5% relative increase. To partially investigate this discrepancy, we quantified collagen volume fraction (CVF) in post-mortem heart tissues of six canines sacrificed at different disease durations (0-22 months). CVF quantified by histology increased from 0.9 to 1.9% (p = 0.56), which agrees better with ECV than with post-contrast myocardial T1 . This study shows that post-contrast myocardial T1 and ECV may disagree in a longitudinal canine study. A more comprehensive study, including histologic, cardiac, and renal functional analyses, is warranted to test rigorously which CMR parameter (ECV or post-contrast myocardial T1 ) agrees better with CVF.

Cervical Human Papillomavirus Testing: Current and Future Impact on Patient Care.

Cervical cancer is the fourth most common malignancy in women worldwide. The identification of human papillomavirus (HPV) as the main etiologic cause of cervical cancer has led to the development and adaptation of HPV molecular diagnostics as a cervical cancer screening and prevention tool. This article highlights six Food and Drug Administration-approved HPV molecular platforms, each with unique advantages and disadvantages. In addition, HPV vaccination and the emergence of HPV self-collection as an alternative testing strategy are discussed.

Molecular signature incorporating the immune microenvironment enhances thyroid cancer outcome prediction.

Genomic and transcriptomic analysis has furthered our understanding of many tumors. Yet, thyroid cancer management is largely guided by staging and histology, with few molecular prognostic and treatment biomarkers. Here, we utilize a large cohort of 251 patients with 312 samples from two tertiary medical centers and perform DNA/RNA sequencing, spatial transcriptomics, and multiplex immunofluorescence to identify biomarkers of aggressive thyroid malignancy. We identify high-risk mutations and discover a unique molecular signature of aggressive disease, the Molecular Aggression and Prediction (MAP) score, which provides improved prognostication over high-risk mutations alone. The MAP score is enriched for genes involved in epithelial de-differentiation, cellular division, and the tumor microenvironment. The MAP score also identifies aggressive tumors with lymphocyte-rich stroma that may benefit from immunotherapy. Future clinical profiling of the stromal microenvironment of thyroid cancer could improve prognostication, inform immunotherapy, and support development of novel therapeutics for thyroid cancer and other stroma-rich tumors.

Simulated fine-needle aspiration diagnosis of follicular thyroid nodules by hyperspectral Raman microscopy and chemometric analysis.

SIGNIFICANCE: Follicular thyroid carcinoma carries a substantially poor prognosis due to its unique biological behavior and less favorable outcomes. In particular, fine-needle aspiration (FNA) biopsies, which play a key role in screening thyroid nodules, cannot differentiate benign from malignant follicular neoplasm. AIM: We report on the use of hyperspectral Raman microscopy in combination with chemometric analysis for identifying and classifying single cells obtained from clinical samples of human follicular thyroid neoplasms. APPROACH: We used a method intended to simulate the FNA procedure to obtain single cells from thyroid nodules. A total of 392 hyperspectral Raman images of single cells from follicular thyroid neoplasms were collected. RESULTS: Malignant cells were identified based on their intrinsic Raman spectral signatures with an overall diagnostic accuracy of up to 83.7%. CONCLUSIONS: Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an ancillary test for analyzing single cells from thyroid FNA biopsies to better stratify "indeterminate" nodules and other cytologically challenging cases.

Multi-institutional validation of a modified scheme for subcategorizing salivary gland neoplasm of uncertain malignant potential (SUMP).

BACKGROUND: The salivary gland neoplasm of uncertain malignant potential (SUMP) category in the Milan System is diagnostically challenging. This study aims to validate a modified scheme for subcategorizing SUMP in a large multi-institutional cohort. METHODS: Retrospective review of salivary gland fine-needle aspirations (FNAs) from 10 institutions were classified based on the Milan System. Cases diagnosed as SUMP with available cytology slides and surgical follow-up were retrieved for review and subcategorized based on a modified scheme as follows: basaloid SUMP (B1: absent/scant nonfibrillary matrix; B2: presence of nonfibrillary/mixed-type matrix), oncocytic/oncocytoid SUMP (O1: with mucinous background; O2: without mucinous background), and SUMP not otherwise specified (NOS). RESULTS: A total of 742 (7.5%) cases from 9938 consecutive salivary gland FNAs were classified as SUMP. Among them, 525 (70.8%) had surgical follow-up and 329 (62.7%) were available for review. The overall risk of malignancy (ROM) of SUMP was 40.4%. There were 156 cases (47.4%) subcategorized as basaloid SUMP with a ROM of 36.5%, 101 (30.7%) as oncocytic/oncocytoid SUMP with a ROM of 52.5%, and 72 (21.9%) as SUMP NOS with a ROM of 31.9%. The ROM of oncocytic/oncocytoid SUMP was significantly higher than basaloid SUMP (P = .0142) and SUMP NOS (P = .0084). No significant differences in ROM were noted between B1 and B2 (36.7% vs 36.4%, P = 1.0000) and O1 and O2 (65.2% vs 48.7%, P = .2349). CONCLUSIONS: The ROM of oncocytic/oncocytoid SUMP was 52.5% and significantly higher than that of basaloid SUMP (36.5%, P = .0142) and SUMP NOS (31.9%, P = .0084), whereas no significant differences in ROM were noted for cases with different types of extracellular matrix or background material.

Validating cell-free DNA from supernatant for molecular diagnostics on cytology specimens.

BACKGROUND: Cytology specimens are often used for biomarker testing in the setting of neoplasia. On occasion, formalin-fixed paraffin-embedded (FFPE) cell blocks unfortunately may not yield sufficient material for testing. Recent studies have suggested that residual supernatant fluid from cell block preparation is a valuable source of DNA: both cellular and cell-free DNA (cfDNA). In the present study, the use of cfDNA from supernatant is compared against DNA from FFPE materials. METHODS: cfDNA was extracted prospectively from residual supernatants of 30 cytology samples (29 neoplastic cases and 1 benign ascitic fluid from a patient with a history of melanoma). Samples were tested using clinically validated next-generation-sequencing platforms and the results were compared with data from paired FFPE cell blocks in a real-time prospective clinical setting. Thirteen samples were tested on an amplicon-based assay (Solid Tumor Hotspot), and 17 samples were tested using a comprehensive capture-based assay (UW-Oncoplex). RESULTS: Neoplastic content was estimated by mutational variant allele fraction, with a mean content of 24.0% and 25.8% in supernatant and FFPE, respectively. The variant concordance between paired samples was 90%, and identical results were detected in both supernatant and FFPE samples in 74% of cases. CONCLUSIONS: This study confirmed that cfDNA from supernatant is a viable alternative to FFPE cell blocks for molecular biomarker testing using both amplicon-based and capture-based assays with potential for decreasing additional tissue sampling and faster turnaround time.

Clinical outcome in diagnostically ambiguous foci of 'gland crowding' in the endometrium.

Premalignant endometrial lesions (endometrial intraepithelial neoplasia (EIN)) are clonal neoplasms that arise focally and can be diagnosed using specific criteria: (1) area of glands exceeds that of stroma (glands/stroma >1), (2) nuclear and/or cytoplasmic features of epithelial cells differ between architecturally abnormal glands and normal background glands, and (3) maximum linear dimension exceeds 1 mm. However, localized groups of crowded endometrial glands may be encountered that do not fulfill all of the criteria for EIN, are interpreted as ambiguous, and are reported as 'focal gland crowding'. We conducted a retrospective study of gland crowding using a free-text index search for this term in our pathology files. The age of the patients, number of subsequent specimens, the duration, and the outcome of the follow-ups were recorded. Of the 71,579 consecutive gynecological pathology reports, 206 (0.3%) 'gland crowding' cases were identified, in which 69% (143/206) had follow-up sampling. Of these, 33 (23%) had an outcome diagnosis of EIN (27 cases; 19%) or carcinoma (6 cases; 4%). Included were 18 cases (55%) diagnosed within the first year and presumed concurrent, and an additional 15 (45%) discovered after 1 year and interpreted as a later phase of disease or new events. The term 'crowded glands' is a highly significant finding that carries a substantial risk of an outcome of EIN and occasionally malignancy. It underscores the importance of follow-up when some but not all of the criteria for EIN are encountered in the appropriate clinical setting.

Raman-based cytopathology: an approach to improve diagnostic accuracy in medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a rare form of thyroid malignancy that can be diagnostically challenging on fine needle aspiration (FNA) cytology. Ancillary tests such as elevated serum or immunohistochemical positive calcitonin have been helpful, yet they can occasionally provide false positive results. In search for an alternative method to improve diagnostic accuracy (DA), we applied hyperspectral Raman spectroscopy to characterize the biochemical composition of single cells from MTC and compared their spectral information to cells from other types of thyroid nodules. Hyperspectral Raman images of 117 MTC single cells from digested tissue were obtained with a line-scan hyperspectral Raman microscope and compared to 127 benign and 121 classic variant of papillary thyroid carcinoma (CVPTC) cells. When principal component analysis and linear discriminant analysis were used to classify the spectral data, MTC cells were differentiated from benign and CVPTC cells with 97% and 99% DA, respectively. In addition, MTC cells exhibited a prominent Raman peak at 1003 cm-1, whose intensity is 84% and 226% greater on average than that observed in benign and CVPTC cells, respectively. When specifically utilizing only this peak as a spectral marker, MTC cells were separated from benign and CVPTC cells with 87% and 95% DA, respectively. As this peak is linked to phenylalanine, which is known to be associated with calcitonin release in thyroid parafollicular cells, the increased intensity further suggests that this Raman peak could potentially be a new diagnostic marker for MTC. Furthermore, preliminary data from MTC cells (n=21) isolated from a simulated FNA procedure provided similar Raman signatures when compared to single cells from digestion. These results suggest that "Raman-based cytopathology" can be used as an adjunct technique to improve the diagnostic accuracy of FNA cytopathology at a single cell level.

Moving forward-the 2019 ASCCP Risk-Based Management Consensus Guidelines for Abnormal Cervical Cancer Screening Tests and Cancer Precursors and beyond: implications and suggestions for laboratories.

The 2019 ASCCP Risk Based Management Consensus Guidelines for prevention of cervical cancer promote clinical management recommendations aligned with our increased understanding of HPV biology and cervical carcinogenesis. They employ HPV-based testing as the basis for risk estimation, allow for personalized risk-based management by incorporating knowledge of current results with prior results, and streamline incorporation of new test methods as they are validated. They continue to support the principles of "equal management for equal risk" and "balancing harms and benefits" adopted in the 2012 version of the guidelines. These updated guidelines will be able to adjust for decreasing CIN3+ risks as more patients who received HPV vaccination reach screening age. Pathology organizations were closely involved in the development of these guidelines. Herein the pathologists who served as representatives to the 2019 ASCCP guidelines steering committee and workgroups, summarize the changes that are relevant to laboratories, pathologists, and cytotechnologists. Prior relevant screening and reporting recommendations that have not been widely and/or inconsistently adopted by laboratories are also discussed and considerations for modification of laboratory practices offered.

Hyperspectral Raman microscopy can accurately differentiate single cells of different human thyroid nodules.

We report on the use of line-scan hyperspectral Raman microscopy in combination with multivariate statistical analyses for identifying and classifying single cells isolated from clinical samples of human thyroid nodules based on their intrinsic Raman spectral signatures. A total of 248 hyperspectral Raman images of single cells from benign thyroid (n = 127) and classic variant of papillary carcinoma (n = 121) nodules were collected. Spectral differences attributed to phenylalanine, tryptophan, proteins, lipids, and nucleic acids were identified for benign and papillary carcinoma cells. Using principal component analysis and linear discriminant analysis, cells were identified with 97% diagnostic accuracy. In addition, preliminary data of cells from follicular adenoma (n = 20), follicular carcinoma (n = 25), and follicular variant of papillary carcinoma (n = 18) nodules suggest the feasibility of further discrimination of subtypes. Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an objective approach for analyzing single cells from fine needle aspiration (FNA) biopsies to enable the minimally invasive diagnosis of "indeterminate" thyroid nodules and other challenging cases.

Sclerosing mucoepidermoid carcinoma with eosinophilia: Cytologic characterization of a rare distinct entity in the thyroid.

Sclerosing mucoepidermoid carcinoma with eosinophilia (SMECE) is an extremely rare thyroid carcinoma with limited cytologic descriptions in the literature. Here, we present a 52-year-old woman with a 3.9 cm thyroid nodule. Fine-needle aspiration smears consisted of a highly cellular specimen with tumor cells in isolated patterns and solid squamoid nests. Tumor cells had round to oval nuclei, prominent nucleoli, smooth nuclear contours, and moderate amounts of dense cytoplasm. In addition to the polymorphous population of lymphocytes, the background contained a striking abundance of eosinophils. The subsequent right thyroidectomy showed histologic features diagnostic for SMECE.

Adventures in Atropisomerism: Total Synthesis of a Complex Active Pharmaceutical Ingredient with Two Chirality Axes.

A strategy to prepare compounds with multiple chirality axes, which has led to a concise total synthesis of compound 1A with complete stereocontrol, is reported.

Accommodative microfluctuations and iris contour.

Mechanical interaction between aqueous humor, iris, and intraocular structures can alter the iris profile from its normal curvature. In particular, significant changes to the iris profile occur during accommodation as the anterior lens movement forces the iris into greater posterior bowing. We extended a previous mathematical model of the anterior segment and investigated the response of this coupled fluid-solid system due to accommodative microfluctuations. The results showed that the system response exhibited the same waveform as the stimulus for small-amplitude microfluctuations generally associated with the high-frequency component. Low-frequency microfluctuations with relatively larger amplitudes elicited a response different from the stimulus, indicating that the forces generated by the lens movement significantly affected the aqueous-iris mechanical interaction.

Immunoisolation of pancreatic epithelial cells from endoscopic ultrasound-guided fine needle aspirates with magnetic beads for downstream molecular application.

BACKGROUND: Evaluation of pancreatic mass is routinely performed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). However, molecular analyses of the tumor cells on FNA samples are limited by the significant cellular heterogeneity. The goal of the current study is to evaluate a magnetic immunoconcentration technique in isolating pancreatic epithelial cells from needle aspirates, and to demonstrate that the isolated cells could be utilized for molecular analysis. METHODS: Pancreatic EUS-FNA specimens were processed and stored at -80°C. Based on the cytopathological diagnosis, 17 adenocarcinoma, 3 lymphoproliferative, and 3 benign cases were retrieved from the collection for further analyses. Epithelial cells were isolated using antihuman epithelial cell specific antibody-bound magnetic beads, and the isolated cellular component was confirmed cytologically. Genomic DNA was extracted, quantitated, and evaluated with methylation-specific PCR for cyclin D2 in 8 adenocarcinoma cases. RESULTS: After optimization, malignant epithelial cells were successfully isolated from all adenocarcinoma cases. Normal pancreatic ductal cells were isolated from three benign cases. No cells were retrieved after immunomagnetic isolation in all three lymphoproliferative cases. DNA yields were 5-2646 ng, with a mean of 357 ng. Methylation-specific PCR for cyclin D2 on the 8 carcinoma cases showed methylated state at the promoter region, demonstrating feasible evaluation of the methylation status. CONCLUSION: The magnetic immunoconcentration of pancreatic EUS-FNA specimen described here is a practical method of isolating pancreatic epithelial cells from needle aspirates. Isolated cells were sufficient for performing subsequent molecular analysis.