The Diagnostic Yield of Malignancy Comparing Cytology, FISH, and Molecular Analysis of Cell Free Cytology Brush Supernatant in Patients With Biliary Strictures Undergoing Endoscopic Retrograde Cholangiography (ERC): A Prospective Study.

BACKGROUND: Routine cytology of biliary stricture brushings obtained during endoscopic retrograde cholangiopancreatography (ERCP) has suboptimal sensitivity for malignancy. We compared the individual and combined ability of cytology, fluorescence in situ hybridization (FISH) analysis and PCR-based mutation profiling (MP) to detect malignancy in standard biliary brushings. METHODS: We performed a prospective study of patients undergoing ERCP using histology or 1 year follow-up to determine patient outcomes. MP was performed on free-DNA from biliary brushing specimens using normally discarded supernatant fluid. MP examined KRAS point mutations and tumor suppressor gene associated loss of heterozygosity mutations at 10 genomic loci. FISH examined chromosome specific gains or losses. RESULTS: A total of 101 patients were included in final analysis and 69% had malignancy. Cytology had 26% sensitivity and 100% specificity for malignancy. Using either FISH or MP in combination with cytology increased sensitivity to 44% and 56%, respectively. The combination of all 3 tests (cytology, FISH, and MP) had the highest sensitivity for malignancy (66%). There was no difference in the specificity of cytology, FISH or MP testing when examined alone or in combination. MP improved diagnostic yield of each procedure from 22% to 100%; FISH improved yield to 90%. MP detected 21 malignancies beyond that identified by cytology; FISH detected an additional 13. The combination of FISH and MP testing detected an additional 28 malignancies. CONCLUSIONS: Both MP and FISH are complimentary molecular tests that can significantly increase detection of biliary malignancies when used in combination with routine cytology of standard biliary brush specimens.

Mutation Profile and Fluorescence In Situ Hybridization Analyses Increase Detection of Malignancies in Biliary Strictures.

BACKGROUND & AIMS: It is a challenge to detect malignancies in biliary strictures. Various sampling methods are available to increase diagnostic yield, but these require additional procedure time and expertise. We evaluated the combined accuracy of fluorescence in situ hybridization (FISH) and polymerase chain reaction-based DNA mutation profiling (MP) of specimens collected using standard brush techniques. METHODS: We performed a prospective study of 107 consecutive patients treated for biliary strictures by endoscopic retrograde cholangiopancreatography from June 2012 through June 2014. We performed routine cytology and FISH analyses on cells collected by standard brush techniques, and analyzed supernatants for point mutations in KRAS and loss-of-heterozygosity mutations in tumor-suppressor genes at 10 loci (MP analysis was performed at Interpace Diagnostics). Strictures were determined to be nonmalignant based on repeat image analysis or laboratory test results 12 months after the procedure. Malignant strictures were identified based on subsequent biopsy or cytology analyses, pathology analyses of samples collected during surgery, or death from biliary malignancy. We determined the sensitivity and specificity with which FISH and MP analyses detected malignancies using the exact binomial test. RESULTS: Our final analysis included 100 patients; 41% had biliary malignancies. Cytology analysis identified patients with malignancies with 32% sensitivity and 100% specificity. Addition of FISH or MP results to cytology results increased the sensitivity of detection to 51% (P < .01) without reducing specificity. The combination of cytology, MP, and FISH analyses detected malignancies with 73% sensitivity (P < .001). FISH identified an additional 9 of the 28 malignancies not detected by cytology analysis, and MP identified an additional 8 malignancies. FISH and MP together identified 17 of the 28 malignancies not detected by cytology analysis. CONCLUSIONS: Addition of FISH and mutation analyses to cytology analysis significantly increased the level of sensitivity with which we detected malignancy in biliary strictures, with 100% specificity. These techniques can be performed using standard brush samples collected during endoscopic retrograde cholangiopancreatography, with mutations detected in free DNA in supernatant fluid of samples. The tests are complementary and therefore should be used sequentially in the diagnostic evaluation of biliary strictures.

The value of mutational profiling of the cytocentrifugation supernatant fluid from fine-needle aspiration of pancreatic solid mass lesions.

Fine-needle aspiration (FNA) of pancreatic solid masses can be significantly impacted by sampling variation. Molecular analysis of tumor DNA can be an aid for more definitive diagnosis. The aim of this study was to evaluate how molecular analysis of the cell-free cytocentrifugation supernatant DNA can help reduce sampling variability and increase diagnostic yield. Twenty-three FNA smears from pancreatic solid masses were performed. Remaining aspirates were rinsed for preparation of cytocentrifuged slides or cell blocks. DNA was extracted from supernatant fluid and assessed for DNA quantity spectrophotometrically and for amplifiability by quantitative PCR (qPCR). Supernatants with adequate DNA were analyzed for mutations using PCR/capillary electrophoresis for a broad panel of markers (KRAS point mutation by sequencing, microsatellite fragment analysis for loss of heterozygosity (LOH) of 16 markers at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q). In selected cases, microdissection of stained cytology smears and/or cytocentrifugation cellular slides were analyzed and compared. In all, 5/23 samples cytologically confirmed as adenocarcinoma showed detectable mutations both in the microdissected slide-based cytology cells and in the cytocentrifugation supernatant. While most mutations detected were present in both microdissected slides and supernatant fluid specimens, the latter showed additional mutations supporting greater sensitivity for detecting relevant DNA damage. Clonality for individual marker mutations was higher in the supernatant fluid than in microdissected cells. Cytocentrifugation supernatant fluid contains levels of amplifiable DNA suitable for mutation detection and characterization. The finding of additional detectable mutations at higher clonality indicates that supernatant fluid may be enriched with tumor DNA. Molecular analysis of the supernatant fluid could serve as an adjunct method to reduce sampling variability and increase diagnostic yield, especially in cases with a high clinical suspicion for malignancy and limited number of atypical cells in the smears.

Alterations in cyst fluid genetics following endoscopic ultrasound-guided pancreatic cyst ablation with ethanol and paclitaxel.

BACKGROUND AND STUDY AIMS: Endoscopic ultrasound (EUS)-guided ethanol lavage with paclitaxel injection has been shown to be effective for the treatment of pancreatic cystic neoplasms; however, the evidence for effectiveness is based primarily on cyst resolution on imaging. The aim of this study was to evaluate changes in pancreatic cyst fluid DNA following EUS-guided pancreatic cyst ablation (PCA) with ethanol and paclitaxel. PATIENTS AND METHODS: In a single-center, prospective study, patients with suspected benign pancreatic cysts (15 - 50 mm in diameter; ≤ 5 compartments) underwent EUS-PCA with ethanol and paclitaxel followed 3 months later by repeat EUS-FNA, cyst aspiration for repeat DNA analysis, and possible repeat EUS-PCA. Abdominal imaging was repeated 3 - 4 months and 12 months after the second EUS. Changes in baseline pancreatic cyst fluid DNA, procedural complications, and radiographic changes in cyst volume were evaluated. RESULTS: A total of 22 patients (median age 67 years; 15 women) with cysts in the head or uncinate (n = 10), body or neck (n = 8), and tail (n = 4), measuring a median diameter of 25 mm (range 15 - 43 mm), underwent one (n = 22) or two (n = 9) EUS-PCA procedures. Baseline cyst DNA included mutations in 11 patients (50 %). Postablation cyst fluid (n = 19) showed elimination of all baseline mutations in eight patients, new mutations in three, and no changes in eight without a baseline mutation. The largest per-protocol postablation image-defined volume change (n = 20) from either of the follow-up abdominal imaging studies (n = 20) demonstrated complete response ( < 5 % original volume) in 10 patients (50 %), partial response (5 % - 25 % original volume) in 5 (25 %), and a persistent cyst (> 25 % original volume) in 5 (25 %). During a median follow-up of 27 months (range 17 - 42 months), adverse events from all EUS-PCAs (n = 31) included abdominal pain alone in four patients (13 %), pancreatitis in three (10 %), peritonitis in one (3 %), and gastric wall cyst in one (3 %). The adverse events were classified as moderately severe in four patients (three with pancreatitis, one with peritonitis). CONCLUSION: EUS-PCA with ethanol and paclitaxel may possibly eliminate mutant DNA in neoplastic pancreatic cysts. This technique leads to complete or partial image-defined resolution in 75 % of cysts but may lead to rare adverse events. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NCT01643460).

The added value of using mutational profiling in addition to cytology in diagnosing aggressive pancreaticobiliary disease: review of clinical cases at a single center.

BACKGROUND: This study aimed to better understand the supporting role that mutational profiling (MP) of DNA from microdissected cytology slides and supernatant specimens may play in the diagnosis of malignancy in fine-needle aspirates (FNA) and biliary brushing specimens from patients with pancreaticobiliary masses. METHODS: Cytology results were examined in a total of 30 patients with associated surgical (10) or clinical (20) outcomes. MP of DNA from microdissected cytology slides and from discarded supernatant fluid was analyzed in 26 patients with atypical, negative or indeterminate cytology. RESULTS: Cytology correctly diagnosed aggressive disease in 4 patients. Cytological diagnoses for the remaining 26 were as follows: 16 negative (9 false negative), 9 atypical, 1 indeterminate. MP correctly determined aggressive disease in 1 false negative cytology case and confirmed a negative cytology diagnosis in 7 of 7 cases of non-aggressive disease. Of the 9 atypical cytology cases, MP correctly diagnosed 7 as positive and 1 as negative for aggressive disease. One specimen that was indeterminate by cytology was correctly diagnosed as non-aggressive by MP. When first line malignant (positive) cytology results were combined with positive second line MP results, 12/21 cases of aggressive disease were identified, compared to 4/21 cases identified by positive cytology alone. CONCLUSIONS: When first line cytology results were uncertain (atypical), questionable (negative), or not possible (non-diagnostic/indeterminate), MP provided additional information regarding the presence of aggressive disease. When used in conjunction with first line cytology, MP increased detection of aggressive disease without compromising specificity in patients that were difficult to diagnose by cytology alone.

The role of the ThyGeNEXT oncogene panel used in combination with the expanded miRNA panel ThyraMIRv2 in Indeterminate thyroid nodules: A large, blinded, real-world, observational study.

BACKGROUND: Molecular analysis of fine-needle aspiration biopsies (FNAB) improves the diagnostic accuracy of cytologically indeterminate thyroid nodules (ITNs). Recently, the use of MPTXv2 has been shown to further improve the accuracy of risk stratification of ITNs. METHODS: A total of 338 patient samples with atypia of undetermined significance (n = 260) or follicular neoplasm (n = 78) cytology diagnosis and corresponding surgical outcomes or clinical follow-up, collected between 2016 and 2020 were included. All samples underwent multiplatform testing (MPTXv1), which includes an oncogene panel (ThyGeNEXT®) plus a microRNA risk classifier (ThyraMIR®). A blinded, secondary analysis was performed to assess the added utility of MPTXv2 (ThyraMIR®v2). The average length of follow-up for the surveillance group (n = 248) was 30 months. RESULTS: Sensitivity at moderate threshold was 96% and specificity at positive threshold was 99% for MPTXv2. At 14% disease prevalence, the negative predictive value at the moderate threshold was 99% and the positive predictive value at the positive threshold was 89% for MPTXv2. MPTXv2 had fewer patients classified into the moderate-risk group than MPTXv1, which was statistically significant (p < .001). Using surgical resection, the gold standard for outcomes, MPTXv2 showed a statistically greater area under the curve (p = .028) than MPTXv1, demonstrating greater accuracy for MPTXv2. CONCLUSION: Both test versions demonstrated robust performance with low false-positive molecular results. Data suggest that incorporation of MPTXv1, and more recently MPTXv2, into clinical practice within our healthcare network resulted in improved accuracy of ITN risk stratification.

Synchronous thyroid cancer and malignant struma ovarii: concordant mutations and microRNA profile, discordant loss of heterozygosity loci.

BACKGROUND: Struma ovarii is an unusual ovarian teratoma containing predominantly thyroid tissue. Less than 10% of cases undergo malignant transformation in the thyroid tissue and are considered malignant struma ovarii (MSO). MSO have been reported with concurrent thyroid lesions, but molecular data is lacking. CASE PRESENTATION: A 42-year-old female developed MSO and synchronous multifocal subcentimeter papillary thyroid carcinoma (PTC). The patient underwent a salpingo-oophrectomy, thyroidectomy, and low-dose radioactive iodine ablation. Both the thyroid subcentimeter PTC and MSO were positive for BRAF V600E mutation, and microRNA expression profiles were similar across all tumor deposits. However, only the malignant component demonstrated extensive loss of heterozygosity (LOH) involving multiple tumor suppressor gene (TSG) chromosomal loci. CONCLUSIONS: We present the first reported case of MSO with synchronous multifocal subcentimeter PTC in the thyroid containing concordant BRAF V600E mutations and resulting with discordant LOH findings. This data suggests that loss of expression in tumor suppressor gene(s) may be an important contributor to phenotypic expression of malignancy.

Molecular analysis of centrifugation supernatant fluid from pancreaticobiliary duct samples can improve cancer detection.

OBJECTIVE: We aimed to supplement microscopic examination of biliary cytobrush specimens to improve sensitivity by mutational profiling of: (1) selected cells microdissected from cytology slides and (2) corresponding cell-free DNA in residual supernatant fluid. STUDY DESIGN: From 43 patients with brushings of bile or pancreatic duct strictures, DNA was extracted from microdissected cells and 1-2 ml of cytocentrifugation supernatant fluid. Mutational analysis targeted 17 genomic sites associated with pancreaticobiliary cancer, including sequencing for KRAS point mutation and loss of heterozygosity (LOH) analysis of microsatellites located at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q. RESULTS: Mutations were found in 25/28 patients with malignancy, and no mutations were found in 5/5 patients with benign surgical results. The cell-free supernatant fluid generally contained higher levels and quality of DNA, resulting in increased detection of mutations in most patients. KRAS mutations only occurred in patients with pancreatic cancer. Mutational profiling of supernatant fluid specimens resulted in high sensitivity and specificity for malignancy, improving the detection of malignancy over cytology alone. CONCLUSION: Brush cytology specimens yielded supernatant fluid enriched with DNA, probably from actively proliferating cells. Mutational profiling can enhance the cytologic evaluation and characterization of specimens suspected to contain pancreatic or bile duct cancer.

A Retrospective Evaluation of the Diagnostic Performance of an Interdependent Pairwise MicroRNA Expression Analysis with a Mutation Panel in Indeterminate Thyroid Nodules.

Background: The addition of genetic analysis to the evaluation of thyroid nodule fine-needle aspiration biopsy samples improves diagnostic accuracy of cytologically indeterminate thyroid nodules (ITNs) with Bethesda III or IV cytopathology. We previously reported the performance of a multiplatform molecular test, referred to in this study as MPTXv1, that includes a mutation panel (ThyGeNEXT®) plus an algorithmic microRNA (miRNA) risk classifier (ThyraMIR®). Complex interactions of growth-promoting and -suppressing miRNAs affect the phenotype. We previously demonstrated that accounting for these interactions with pairwise miRNA expression analysis improves the diagnosis of medullary thyroid carcinoma. In this study, we assess the impact of pairwise miRNA expression analysis on risk stratification of ITNs. Methods: Pairwise expression analysis of 11 miRNAs was performed on a training cohort of histopathology-proven benign nodules (n = 50) to define the mean and standard deviation of each pairwise analysis and create a Benign/Malignant Profiler (MPTXv2), deviations from which predicted the malignancy risk. Clinical validation of MPTXv2 was assessed using a cohort of 178 ITN (Bethesda III and IV) samples from a multicentered, blinded retrospective study, previously evaluated by MPTXv1. Results: Compared with MPTXv1, MPTXv2 significantly improved the test performance. The receiver operating characteristic (ROC) areas under the curve (AUC) increased from 0.85 to 0.97 (p < 0.001), and the diagnostic accuracy at the positive threshold increased significantly (p < 0.05) from 83% [95% confidence interval (CI) = 76-88] to 93% [CI = 89-96]. The significant improvement in the ROC AUC and the diagnostic accuracy was due to a strong statistical trend for improvement in specificity at the positive threshold. At the positive threshold, the specificity for MPTXv1 was 90% [CI = 84-95] and improved to 98% [CI = 94-99] for MPTXv2. Using the MPTXv2, the Moderate-Risk cohort decreased from 50 samples (28% of the cohort) to 24 samples (13% of the cohort). This 52% decrease is statistically significant (p < 0.001) and clinically meaningful. Conclusion: As compared with MPTXv1, pairwise miRNA expression analysis used in MPTXv2 significantly improved the diagnostic accuracy of ITN risk stratification and reduced the size of the Moderate-Risk group. Prospective trials are indicated to confirm these findings in a clinical practice setting.

Analytical and clinical validation of pairwise microRNA expression analysis to identify medullary thyroid cancer in thyroid fine-needle aspiration samples.

BACKGROUND: Medullary thyroid carcinoma (MTC) is an aggressive malignancy originating from the parafollicular C cells. Preoperatively, thyroid nodule fine-needle aspiration cytology (FNAC) and pathogenic gene mutations are definitive in approximately one-half of cases. MicroRNAs (miRNAs) are endogenous, noncoding, single-stranded RNAs that regulate gene expression, a characteristic that confers the potential for identifying malignancy. In the current study, the authors hypothesized that differential pairwise (diff-pair) analysis of miRNA expression levels would reliably identify MTC in FNA samples. METHODS: The relative abundance of 10 different miRNAs in total nucleic acids was obtained from ThyraMIR test results. Diff-pair analysis was performed by subtracting the critical threshold value of one miRNA from the critical threshold values of other miRNAs. Next-generation sequencing with the ThyGeNEXT panel identified oncogenic gene alterations. The discovery cohort consisted of 30 formalin-fixed, paraffin-embedded benign and malignant thyroid neoplasms, including 4 cases of MTC. After analytical validation, clinical validation was performed using 3 distinct cohorts (total of 7557 specimens). RESULTS: In the discovery cohort, 9 diff-pairs were identified as having significant power using the Kruskal-Wallis test (P < .0001) to distinguish MTC samples from non-MTC samples. The assay correctly classified all MTC and non-MTC samples in the analytical validation study and in the 3 clinical validation cohorts. The overall test accuracy was 100% (95% confidence interval, 99%-100%). In indeterminate FNAC samples, the sensitivity of the diff-pair analysis was greater than that of the MTC-specific mutation analysis (100% vs 25%; P = .03). CONCLUSIONS: Pairwise miRNA expression analysis of ThyraMIR results were found to accurately predict MTC in thyroid FNA samples, including those with indeterminate FNAC findings.

Incremental utility of expanded mutation panel when used in combination with microRNA classification in indeterminate thyroid nodules.

INTRODUCTION: Focused and expanded mutation panels were assessed for the incremental utility of using an expanded panel in combination with microRNA risk classification. METHODS: Molecular results were reviewed for patients who underwent either a focused mutation panel (ThyGenX®) or an expanded mutation panel (ThyGeNEXT®) for strong and weak oncogenic driver mutations and fusions. microRNA results (ThyraMIR®) predictive of malignancy, including strong positive results highly specific for malignancy, were examined. RESULTS: Results of 12 993 consecutive patients were reviewed (focused panel = 8619, expanded panel = 4374). The expanded panel increased detection of strong drivers by 8% (P < .001), with BRAFV600E and TERT promoters being the most common. Strong drivers were highly correlated with positive microRNA results of which 90% were strongly positive. The expanded panel increased detection of coexisting drivers by 4% (P < .001), with TERT being the most common partner often paired with RAS. It increased the detection of weak drivers, with RAS and GNAS being the most common. 49% of nodules with weak drivers had positive microRNA results of which 33% were strongly positive. The expanded panel also decreased the number of nodules lacking mutations and fusions by 15% (P < .001), with 8% of nodules having positive microRNA results of which 22% were strongly positive. CONCLUSIONS: Using expanded mutation panels that include less common mutations and fusions can offer increased utility when used in combination with microRNA classification, which helps to identify high risk of malignancy in the cases where risk is otherwise uncertain due to the presence of only weak drivers or the absence of all drivers.

Development and Analytical Validation of an Expanded Mutation Detection Panel for Next-Generation Sequencing of Thyroid Nodule Aspirates.

Molecular analysis is used to evaluate the risk of malignancy for thyroid fine-needle aspirates, identified as indeterminate by microscopic cytology, on the basis of the detection of various oncogenic DNA mutations and fusion transcripts or on the use of various mRNAs or miRNA-based classifier algorithms. Our approach has been to use a combination test using the detection of oncogenic mutations/fusion transcripts and an miRNA expression-based classifier algorithm. To improve the performance of the combination test, the next-generation sequencing (NGS)-based mutational panel was expanded from the detection of 5 oncogenes to 10 oncogenes and tumor suppressor genes and the detection of fusion transcripts was increased from 6 to 38. Herein, we describe the assay development of the expanded panel NGS test and optimization of various steps for the library preparation of multiplexed target genes to maintain quality parameters for sequencing and to improve the robustness of the test for use in clinical testing in a College of American Pathologists/Clinical Laboratory Improvements Amendments-certified laboratory. Technical hurdles in NGS library preparation for the sequencing of both normal and high guanine-cytosine-rich regions, and balanced amplification of various amplicons in highly multiplexed PCRs, were successfully overcome. Analytical validation as a laboratory-developed test (ThyGeNEXT) included the demonstration of assay reproducibility, lower limit of detection, as well as other fundamental quality parameters.

Multiplatform molecular test performance in indeterminate thyroid nodules.

BACKGROUND: Approximately 25% of thyroid nodule fine-needle aspirates (FNAs) have cytology that is indeterminate for malignant disease. Accurate risk stratification of these FNAs with ancillary testing would reduce unnecessary thyroid surgery. METHODS: We evaluated the performance of an ancillary multiplatform test (MPTX) that has three diagnostic categories (negative, moderate, and positive). MPTX includes the combination of a mutation panel (ThyGeNEXT®) and a microRNA risk classifier (ThyraMIR®). A blinded, multicenter study was performed using consensus histopathology diagnosis among three pathologists to validate test performance. RESULTS: Unanimous consensus diagnosis was reached in 197 subjects with indeterminate thyroid nodules; 36% had disease. MPTX had 95% sensitivity (95% CI,86%-99%) and 90% specificity (95% CI,84%-95%) for disease in prevalence adjusted nodules with Bethesda III and IV cytology. Negative MPTX results ruledout disease with 97% negative predictive value (NPV; 95% CI,91%-99%) at a 30% disease prevalence, while positive MPTX results ruledin high risk disease with 75% positive predictive value (PPV; 95% CI,60%-86%). Such results are expected in four out of five Bethesda III and IV nodules tested, including RAS positive nodules in which the microRNA classifier was useful in rulingin disease. 90% of mutation panel false positives were due to analytically verified RAS mutations detected in benign adenomas. Moderate MPTX results had a moderate rate of disease (39%, 95% CI,23%-54%), primarily due to RAS mutations, wherein the possibility of disease could not be excluded. CONCLUSIONS: Our results emphasize that decisions for surgery should not solely be based on RAS or RAS-like mutations. MPTX informs management decisions while accounting for these challenges.

Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study.

BACKGROUND: The role of pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts remains unclear. OBJECTIVE: Our purpose was to evaluate the utility of a detailed DNA analysis of pancreatic cyst fluid to diagnose mucinous and malignant cysts. DESIGN: Prospective, multicenter study. PATIENTS: Patients with pancreatic cysts presenting for EUS evaluation. INTERVENTION: EUS-guided pancreatic cyst aspirates cytology evaluation, carcinoembryonic antigen (CEA) level determination, and a detailed DNA analysis; incorporating DNA quantification, k-ras mutation and multiple allelic loss analysis, mutational amplitude, and sequence determination. MAIN OUTCOME MEASUREMENTS: Cyst fluid analysis compared with surgical pathologic or malignant cytologic examination. RESULTS: The study cohort consisted of 113 patients with 40 malignant, 48 premalignant, and 25 benign cysts. Cyst fluid k-ras mutation was helpful in the diagnosis of mucinous cysts (odds ratio 20.9, specificity 96%), whereas receiver-operator characteristic curve analysis indicated optimal cutoff points for allelic loss amplitude (area under the curve [AUC] 0.79; optimal value > 65%) and CEA (AUC 0.74; optimal value >148 ng/mL). Components of DNA analysis detecting malignant cysts included allelic loss amplitude over 82% (AUC 0.9) and high DNA amount (optical density ratio >10, AUC 0.79). The criteria of a high amplitude k-ras mutation followed by allelic loss showed maximum specificity (96%) for malignancy. All malignant cysts with negative cytologic evaluation (10/40) could be diagnosed as malignant by using DNA analysis. LIMITATIONS: Limited follow-up, selection bias. CONCLUSIONS: Elevated amounts of pancreatic cyst fluid DNA, high-amplitude mutations, and specific mutation acquisition sequences are indicators of malignancy. The presence of a k-ras mutation is also indicative of a mucinous cyst. DNA analysis should be considered when cyst cytologic examination is negative for malignancy.

Loss of Heterozygosity (LOH) at 17p13 and 22q13 are Shared by Breast and Thyroid Carcinomas for Metastasis.

Many genomic mutations have been identified to be related to the metastasis of malignancies from various primary sites. In this study, we attempted to identify the loss of heterozygosity (LOH) that might be involved in metastasis of breast ductal carcinoma (BDC) and papillary thyroid carcinoma (PTC). We retrieved 14 BDC cases with metastasis and 19 BDC cases without metastasis as well as 12 PTC cases with metastasis and 14 PTC cases without metastasis. Analysis of 13 polymorphic microsatellite repeat markers targeting 1p34-36, 3p24-26, 9p21, 10q23, 17p13, 17q21, 21q22, and 22q13 was performed on DNA isolated from primary tumors. The results showed that LOH at 17p13 and 22q13 was shared by both BDC and PTC for metastasis. More detailed studies to identified genes in these shared loci of LOH may provide further insight into the molecular mechanisms underlying metastases in these 2 tumor types, and possibly other malignancies as well.

Diagnostic and prognostic utility of molecular markers in synchronous bilateral breast carcinoma.

Histologic criteria have a limited role in determining whether the synchronous bilateral breast carcinomas represent two primaries or a metastasis to the contralateral breast. We studied the molecular analysis of synchronous bilateral breast carcinoma and whether they are originating from a single or different clone. We examined 17 patients with breast carcinoma, including 12 patients with synchronous bilateral carcinomas and control group of 5 infiltrating ductal carcinomas with regional lymph node metastases. Mutations were quantitatively determined to detect loss of heterozygosity (LOH) and microsatellite size alterations for a broad panel of 15 markers, involving 10 chromosomes using polymerase chain reaction. The carcinomas were classified as de novo or metastasis based on three levels of concordance: (1) marker-affected tumors were considered concordant if 50% or more of the same markers were mutated, (2) same gene copy affected, and (3) temporal sequence of mutation acquisition. In synchronous bilateral breast carcinoma patients, molecular analysis showed discordant mutations in all cases, supporting the diagnosis of de novo bilateral primary breast carcinomas. In patients with lymph node metastases, the primary breast carcinoma and metastases shared the same mutations, revealing a metastatic lesion. In conclusion, the application of molecular technology may play an important role for the differential diagnosis of dual primary carcinomas vs a metastatic breast cancer to contralateral breast. In this study, synchronous bilateral breast cancers represent two independent primaries rather than metastatic events.

Fractional allelic imbalance could allow for the development of an equitable transplant selection policy for patients with hepatocellular carcinoma.

Liver transplantation (LT) in the presence of hepatocellular carcinoma (HCC) remains a controversial issue because the current staging systems are not sufficiently predictive of outcomes. Paraffin blocks from 183 patients that underwent LT in the presence of HCC were collected. Molecular analysis was carried out blindly on the native liver specimens in all cases with respect to recurrence outcomes. The fractional allelic imbalance (FAI) rate index was determined in each case and was used to compare the acquired mutational load between different tumors. The FAI was determined from the microdissected tissue site displaying the greatest amount of acquired allelic loss. FAI was found to be the strongest predictor of recurrence followed by vascular invasion and then by tumor number or hepatic lobar involvement. Based on these findings, 3 prognostic models were constructed for selection of candidates for LT in patients with concomitant HCC. Molecular markers of tumor progression are the strongest predictors of HCC recurrence currently available, surpassing all components of the tumor-node-metastasis classification system for staging of malignant tumors (TNM), including vascular invasion. Incorporation of these molecular markers of tumor progression could help resolve the ongoing conundrum of organ allocation for patients with HCC.

Expression of p53 and prognosis in children with malignant gliomas.

BACKGROUND: The prognosis of children with high-grade gliomas is uncertain, even when clinical and histologic findings are considered. We investigated whether mutations in the TP53 gene or the degree of expression of p53 protein in high-grade gliomas is associated with progression-free survival in children with these tumors. METHODS: Paraffin-embedded specimens of malignant gliomas from children treated in the Children's Cancer Group study CCG-945 were assessed by mutational analysis of TP53 (121 specimens) and immunohistochemical analysis of p53 (115 specimens). For mutational studies, areas of tissue that contained malignant glioma were isolated by microdissection, and the DNA was subjected to polymerase-chain-reaction-based amplification and sequencing of TP53 exons 5, 6, 7, and 8. Immunohistochemical analysis was performed with the use of a microwave-enhanced antigen retrieval and an antibody that bound both wild-type and mutant p53. RESULTS: We found a significant association between overexpression of p53 and outcome; this association was independent of histologic features, age, sex, the extent of resection, and tumor location. The rate ( +/- SE) of progression-free survival at five years was 44 +/- 6 percent in the group of 74 patients whose tumors had low levels of expression of p53 and 17 +/- 6 percent in the group of 41 patients whose tumors had overexpression of p53 (P<0.001). A nonsignificant association was observed between mutations in TP53 and outcome. CONCLUSIONS: Overexpression of p53 in malignant gliomas during childhood is strongly associated with an adverse outcome, independently of clinical prognostic factors and histologic findings.

A massive hepatic tumor demonstrating hepatocellular, cholangiocarcinoma and neuroendocrine lineages: A case report and review of the literature.

INTRODUCTION: Mixed hepatocellular and cholangiocarcinoma tumors (MHCC) are described in the literature, as are the more rare mixed adenoneuroendocrine carcinomas (MANC) of hepatobiliary origin. Only two cases of tumors with characteristics of all three histologies/phenotypes have been previously described in one Chinese study. PRESENTATION OF CASE: Herein we report clinical, microscopic and molecular features of a 25cm mixed hepatic tumor with hepatocellular, cholangiocarcinoma and neuroendocrine differentiation arising in an otherwise healthy 19-year-old North American Caucasian male without any identifiable risk factors. DISCUSSION: The patient underwent multimodality imaging and the tumor was biopsied preoperatively, and it was initially interpreted to be hepatocellular carcinoma fibrolamellar type. A left trisegmentectomy with lymphadenectomy was performed and the tumor was definitively diagnosed based on the surgically resected specimen. Integrated microscopic and molecular features defined the differing biological aggressiveness of growth pattern components. Cases in the literature of MHCC and rare cases of MANC have largely undergone aggressive surgical resection as well, however the majority of studies on mixed hepatic tumors to date reflect Eastern patient cohorts and populations with underlying liver disease, thereby limiting extrapolation on management or outcomes in this case. CONCLUSION: This is one of the only reports of a hepatic tumor arising from hepatocellular carcinoma, cholangiocarcinoma and neuroendocrine lineages. Increased awareness of this tumor type may optimize improve future management.