Cumulative sum procedure in evaluation of EUS-guided FNA cytology: the learning curve and diagnostic performance beyond sensitivity and specificity

Background: Using cumulative sum (CUSUM) chart, we address two questions: (i) Over time, how will an EUS‐FNA (endoscopic ultrasound guided fine needle aspiration) service maintain an acceptable non‐diagnostic rate defined as technical failures, unsatisfactory specimens and atypical and suspicious diagnoses? (ii) Over time, how will EUS‐FNA maintain acceptable diagnostic errors (false‐positives plus false‐negative diagnosis)? Methods: The study included all consecutive patients who underwent EUS‐FNA at our institution from July 2000 to October 2003 and were followed up until December 2004. Using a simple spread sheet, we designed CUSUM charts and used them to track trends and assess performance at a preset acceptable rate of 10% and a preset unacceptable rate of 15% for non‐diagnostic rate and diagnostic errors. We assessed all cases collectively and then in groups defined by site, size and cytopathologist. Results: Of 876 patients undergoing EUS‐FNA, 83 (9.5%) had non‐diagnostic results: 43 (51%) of these diagnoses were ‘atypical’, 27(33%) were ‘suspicious for malignancy’, eight (10%) were ‘insufficient material for diagnosis’ and five (6%) were ‘technical failure’. In 585 cases with adequate follow up, there were 26 (6.3%) diagnostic errors: three (0.5%) were false positive and 23 (3.1) were false negative. The overall CUSUM charts for both non‐diagnostic rate and for diagnostic error rate start with a small period of learning then cross to a significantly acceptable level at case numbers 121 and 97 respectively. Our diagnostic performance was better in lymph nodes than in the pancreas and other organs and was not significantly different for lesions ≤25 mm compared with lesion >25 mm in diameter. Performance was better for pathologists with prior experience than for pathologists without experience. Conclusion: In the current climate of proficiency testing, error tracking and competence evaluation, there is a great potential for the use of CUSUM charts to assess procedure failure and error tracking in quality control programs, particularly when a new procedure such as EUS‐FNA is introduced in the laboratory. Additionally, the method can be used to assess trainee competency and to track the proficiency of practicing cytologists.     

The application of fine needle aspiration cytology in the diagnosis of multiple primary malignant tumors

Four cases with multiple primary malignant tumors are presented. In all cases, fine needle aspiration (FNA) cytologic findings indicated the presence of more than one primary malignancy. In one case, the cytologic examination simultaneously diagnosed two separate primaries. Since FNA cytology can often be used to identify the tumor type, it can be utilized in the identification of many multiple primary malignancies, as these cases show.     

Multi‐species nitrifying biofilm model (MSNBM) including free ammonia and free nitrous acid inhibition and oxygen limitation

A multi‐species nitrifying biofilm model (MSNBM) is developed to describe nitrite accumulation by simultaneous free ammonia (FA) and free nitrous acid (FNA) inhibition, direct pH inhibition, and oxygen limitation in a biofilm. The MSNBM addresses the spatial gradient of pH with biofilm depth and how it induces changes of FA and FNA speciation and inhibition. Simulations using the MSNBM in a completely mixed biofilm reactor show that influent total ammonia nitrogen (TAN) concentration, bulk dissolved oxygen (DO) concentration, and buffer concentration exert significant control on the suppression of nitrite‐oxidizing bacteria (NOB) and shortcut biological nitrogen removal (SBNR), but the pH in the bulk liquid has a weaker influence. Ammonium oxidation increases the nitrite concentration and decreases the pH, which together can increase FNA inhibition of NOB in the biofilm. Thus, a low buffer concentration can accentuate SBNR. DO and influent TAN concentrations are efficient means to enhance DO limitation, which affects NOB more than ammonia‐oxidizing bacteria (AOB) inside the biofilm. With high influent TAN concentration, FA inhibition is dominant at an early phase, but finally DO limitation becomes more important as TAN degradation and biofilm growth proceed. MSNBM results indicate that oxygen depletion and FNA inhibition throughout the biofilm continuously suppress the growth of NOB, which helps achieve SBNR with a lower TAN concentration than in systems without concentration gradients. Biotechnol. Bioeng. 2010;105: 1115–1130. © 2009 Wiley Periodicals, Inc.     

Interpretation of fine needle aspiration cytology of the breast: a comparison of cytological, frozen section, and final histological diagnoses

This study examines the performance of the preliminary, on-site interpretation by the pathologist of fine needle aspiration (FNA) cytology smears compared to the final cytology report, the frozen section diagnosis and the final histopathological report. We found that both the preliminary and the final cytology reports gave satisfactory results over the minimum standards for quality assurance required by both the Norwegian breast screening programme and the NHS BSP in the UK with the exception of the 'suspicious' rate. We noted that the preliminary report had fewer false negatives (2.1%) than the final report (4.3%). We show that an unequivocal cytological diagnosis of malignancy is a reliable diagnosis, and in cases where mammography/ultrasonography and clinical examination are in agreement with FNA, frozen section examination is unnecessary. However, cases with a suspicious or equivocal FNA should be considered for frozen section analysis.     

Value of Repeating A Nondiagnostic Thyroid Fine-Needle Aspiration Biopsy

ObjectiveTo assess the value of repeating a biopsy when the initial thyroid fine-needle aspiration (FNA) biopsy is nondiagnostic.MethodsBetween 1990 and 2003, 4,311 thyroid FNAs were performed at the Cleveland Clinic Foundation, of which 220 (5%) were nondiagnostic. Among 189 patients whose medical records were available for retrospective review, 106 underwent a repeated FNA (FNA #2), and 14 had a second repeated FNA (FNA #3). Thyroid ultrasonography was used in the evaluation in 113 FNAs.ResultsThe first and second repeated FNAs were diagnostic in 58% (62 of 106 patients) and 50% (7 of 14 patients), respectively. The rate of malignant disease in patients with no repeated FNAs versus 1 or more repeated FNAs was 4.8% (4 of 83) versus 11.3% (12 of 106), respectively. Ultrasound-guided FNA yielded a diagnosis among 33 of 113 biopsies (29.2%), and FNA without ultrasound guidance provided a diagnosis in 30 of 159 biopsies (18.9%). Thus, the use of thyroid ultrasonography significantly improved the likelihood of establishing a diagnosis (P = 0.017). We found that repeating the FNA up to 2 times provides a diagnosis in up to 60% of cases.ConclusionThe overall prevalence of thyroid cancer in patients with nondiagnostic FNA is not trivial—8.5% in our study group of 189 patients. An aggressive approach toward nondiagnostic FNA biopsies is recommended, with performance of at least 2 repeated FNA biopsies, preferably with the help of ultrasound guidance. (Endocr Pract. 2007;13:735-742)     

Free nitrous acid (FNA) inhibition on denitrifying poly-phosphate accumulating organisms (DPAOs)

Free nitrous acid (FNA) has been identified to be a ubiquitous inhibitor of a wide range of microorganisms, including bacteria involved in wastewater treatment. The FNA-induced inhibition on the anoxic (nitrite as electron acceptor) metabolism of denitrifying poly-phosphate accumulating organisms (DPAOs) was investigated using sludge from a sequencing batch reactor performing carbon, nitrogen, and phosphorus removal from synthetic wastewater. We found that FNA had a much stronger inhibitory effect on phosphorus (P) uptake and glycogen production than on poly-β-hydroxyalkanoate degradation and nitrite reduction. The intracellular adenosine triphosphate levels decreased sharply during the FNA incubation, and the decreasing rates were positively correlated with increasing FNA concentrations. The electron transport activity of DPAOs when exposed to FNA displayed a similar trend. Further, at FNA concentrations above 0.044 mg HNO₂-N/L, the anaerobic metabolism of DPAOs was initiated despite of the presence of nitrite, as evidenced by the release of phosphorus and the consumption of glycogen. DPAO metabolism did not recover completely from FNA inhibition in the subsequent FNA-free environment. The recovery rate depended on the concentration of FNA applied in the previous anoxic period. These results suggest that the inhibitory effects are diverse and may be attributable to different mechanisms operating simultaneously.     

Focus: Personalized Medicine: Simultaneously Detection of 50 Mutations at 20 Sites in the BRAF and RAS Genes by Multiplexed Single-Nucleotide Primer Extension Assay Using Fine-Needle Aspirates of Thyroid Nodules

Fine-needle aspiration (FNA) is commonly used for primary evaluation of thyroid nodules. Twenty to 30 percent of thyroid nodules remain indeterminate after FNA evaluation. Studies show the BRAF p.V600E to be highly specific for papillary thyroid carcinoma (PTC), while RAS mutations carry up to 88 percent positive predictive value for malignancy. We developed a two-tube multiplexed PCR assay followed by single-nucleotide primer extension assay for simultaneous detection of 50 mutations in the BRAF (p.V600E, p.K601E/Q) and RAS genes (KRAS and NRAS codons 12, 13, 19, 61 and HRAS 61) using FNA smears of thyroid nodules. Forty-two FNAs and 27 paired formalin-fixed, paraffin-embedded (FFPE) tissues were tested. All BRAF p.V600E-positive FNA smears (five) carried a final diagnosis of PTC on resection. RAS mutations were found in benign as well as malignant lesions. Ninety-two percent concordance was observed between FNA and FFPE tissues. In conclusion, our assay is sensitive and reliable for simultaneous detection of multiple BRAF/RAS mutations in FNA smears of thyroid nodules.     

A decision-analytic framework for impact assessment part I: LCA and decision analysis

Life-cycle assessments (LCAs) are conducted to satisfy the aspiration of decision makers to consider the environment in their decision making. This paper reviews decision analysis and discusses how it can be used to structure the assessment and to integrate characterization and valuation. The decision analytic concepts of objectives (goals) and attributes (indicators of the degree to which an objective is achieved) are used to describe steps of the assessment of the entire impact chain. Decision analysis distinguishes among different types of objectives and attributes; it describes how these relate to each other. Impact indicators such as the Human Toxicity Potential are constructed attributes. A means-ends objectives network can show how the different constructed attributes relate to the objective of protecting the environment. As LCA takes disparate environmental impacts into account, it needs to assess their relative importance. Trade-off methods in decision analysis are grouped into utility theory and multicriteria decision aids; they have different advantages and disadvantages, but are all more sophisticated than simple weighting. The performance of the different trade-off methods has not yet been tested in an LCA context. In the second part of the paper, we present criteria for the development of characterization methods.     

Immunocytochemistry of fine needle aspirates. A tactical approach

To maximize the potential of immunocytodiagnosis for fine needle aspiration (FNA) samples, it is necessary to be aware of the pitfalls and limitations of these techniques and to formulate a strategy to deal with the many variables involved. Five cases are presented to illustrate some of these variables, which include determining the adequacy of the FNA specimen, selecting tactics for cytologic and immunocytochemical studies, selecting methods for processing the FNA sample, preparing smears to enrich and preserve cells of interest, selecting enzyme labeling methods to optimize sensitivity and specificity, selecting monoclonal antibodies to make the study efficient and pertinent and interpreting the study results. The adequacy of the FNA specimen could be determined by an immediate cytologic assessment of the aspirate as it was obtained. Alcohol-fixed smears and formalin-fixed tissue sections prepared from the aspirate were used for diagnosis; the immunocytochemical studies were used as a diagnostic adjunct for accurate cell identification. Immunocytochemical studies were done on air-dried cytocentrifuge smears of pre-washed cells. While both immunoperoxidase and immunoalkaline phosphatase methods were suitable, we recommend the immunoperoxidase method for the study of aspirates from nonhemopoietic tissues and the immunoalkaline phosphatase method for the study of aspirates with many blood cells present. The proper selection of monoclonal antibodies and the interpretation of the results are best made in the context of the cytologic characteristics of the FNA sample and the clinical features of the patient.     

Diagnosis of deep‐seated lymphomas by endoscopic ultrasound‐guided fine needle aspiration combined with flow cytometry

A. Stacchini, P. Carucci, D. Pacchioni, G. Accinelli, A. Demurtas, S. Aliberti, M. Bosco, M. Bruno, A. Balbo Mussetto, M. Rizzetto, G. Bussolati and C. De Angelis
Diagnosis of deep‐seated lymphomas by endoscopic ultrasound‐guided fine needle aspiration combined with flow cytometry Objective: Although endoscopic ultrasound combined with fine needle aspiration (EUS‐FNA) is rapidly becoming the preferred diagnostic approach for the sampling and diagnosis of gastrointestinal and mediastinal malignancies, there are limited data as to its use in the diagnosis of lymphoproliferative disorders. Therefore, we carried out a retrospective evaluation of the performance of EUS‐guided FNA combined with flow cytometry (FC) as a tool to improve overall sensitivity and specificity in the diagnosis of lymphoma. Methods: Of 1560 patients having EUS‐guided FNA during the period of the study, a total of 56 patients were evaluated by cytology with FC after EUS‐FNA. There was adequate material to perform FC analysis for all but one case. Results: EUS‐FNA‐FC gave a diagnosis of lymphoma in 11 cases and of reactive lymphadenopathy in 20. A specific histological type was defined by FC alone in eight cases. The remaining cases were diagnosed later by cytology and cell block sections: 13 carcinomas, nine granulomatous lymphadenopathies and one mediastinal extramedullary haematopoiesis. One case was considered only suspicious for lymphoma on cytology and FC but was not confirmed on molecular analysis and one had insufficient material for FC. Conclusions: Our results show that a combination of EUS‐FNA‐FC is a feasible and highly accurate method, which may be used for the diagnosis and subtyping of deep‐seated lymphoma, providing a significant improvement to cytomorphology alone both for diagnosis and treatment planning, as long as immunocytochemistry is available for non‐lymphoma cases.     

Correlation of Findings from Iodine 123 Scan and Ultrasonography in the Recommendation for Thyroid Fine-Needle Aspiration Biopsy

ObjectiveTo assess the impact of correlating findings from iodine I 123 (¹²³I) radionuclide scans and thyroid ultrasonography on the decision to perform fine-needle aspiration (FNA) biopsy of thyroid nodules.MethodsIodine 123 scans and sonographic images of adult patients who had both examinations performed within 6 months of each other at our institution were retrospectively reviewed. The presence of 1 or more nodules satisfying imaging-specific criteria for recommending FNA biopsy was recorded. Iodine 123 scan and sonographic images were then directly compared to determine how frequently the FNA recommendation would be affected by discordant findings.ResultsThe study included 97 adult patients, with a total of 291 thyroid lobes (right thyroid lobe, left thyroid lobe, and isthmus). Recommendations for FNA biopsy were concordant in 231 of 291 lobes (79.4%), with both modalities recommending FNA biopsy in 55 lobes and not recommending FNA biopsy in 176 lobes. A discordant recommendation occurred in 60 of 291 lobes (20.6%). Using only ultrasonography findings, a recommendation for FNA biopsy was not indicated for 11 of the 291 lobes (3.8%) with functioning nodules. Using only ¹²³I findings, a recommendation for FNA biopsy was not indicated for 23 of the 291 lobes (7.9%); 13 had nodules, but none that fulfilled sonographic criteria, and 10 had no identifiable nodule on ultrasonography. Iodine 123 scan did not identify 26 lobes with nodules (8.9%) for which FNA biopsy was recommended based on ultrasonography findings.ConclusionRecommendations for FNA biopsy should not be based on the presence of hypofunctioning regions on ¹²³I scan without sonographic confirmation. (Endocr Pract. 2011;17:699-706)     

Fine needle aspiration: past,current practice and recent developments

Fine needle aspiration (FNA) is a diagnostic technique that has become widely used. The procedure may be performed by interventional radiologists, endoscopists, pathologists and cytotechnologists; diagnostic interpretation of FNA samples is performed by pathologists and cytotechnologists. I provide here an introduction to the technique and applications of FNA with a review of current practice parameters and recent developments in the molecular application of FNA.     

Value of the cytologic analysis of fine needle aspiration biopsy specimens in the diagnosis of pancreatic carcinomas

Between 1982 and 1986, 410 preoperative percutaneous fine needle aspiration (FNA) biopsies of the pancreas were performed on 316 patients clinically suspected of having a malignant pancreatic tumor. Of 58 patients with pancreatic carcinomas subsequently confirmed by histologic investigation, the FNA biopsy yielded a cytologically positive diagnosis of carcinoma in 39 cases (67.2%) and suspicious findings in another 5 cases (8.6%). In 14 cases of malignancy (24.1%), the FNA puncture failed to sample material from the tumor; hence, the cytologic evaluation yielded false-negative results. Of 21 patients with inflammatory disorders of the pancreas, cytologically suspicious cells were observed in 5 cases (23.8%); in none of those 5 cases did the histologic examination show any evidence of carcinoma. This indicates that caution should be taken not to cytologically over-diagnose cases of pancreatitis. On the whole, cytology proved to be a valuable method for the diagnosis of pancreatic carcinoma; it provided the highest rate of positive results in comparison with other modern clinical diagnostic methods. Furthermore, cytology may improve the diagnostic results even in those cases with clinically negative or merely suspicious findings. FNA punctures of the pancreas produced no serious complications in this series.     

Assessment of fine needle aspiration as a screening test for occult prostatic carcinoma

As part of an ongoing study of objective parameters of prognostic value in prostatic carcinoma, a routine procedure was developed to aspirate all prostates prior to surgery. These targets were different from those of other workers in the field of prostatic fine needle aspiration (FNA), who generally advocate that FNA be confined to suspicious nodules. The aspirations were performed by a large group of practicing urologists who had no special training in prostatic FNA except for guidelines provided by their peers and information available in the literature. This approach permitted an assessment of the performance of FNA as a screening test rather than as a diagnostic procedure. During the period from January 1983 to February 1987, 1,683 patients had prostatic FNAs performed (plus subsequent histologic study). The following diagnoses were rendered: "inadequate/scanty specimen" in 625 cases (37%), "negative/atypical" in 844 cases (50%) and "suspicious/positive" in 214 cases (13%). Histologic examination showed stage A1 prostatic adenocarcinoma in 18 patients. The cytologic diagnoses on these 18 patients were inadequate/scanty in 3 (17%), negative/atypical in 13 (72%) and suspicious/positive in 2 (11%). Of the 214 patients with a positive/suspicious diagnosis by FNA, the diagnosis of prostatic carcinoma was confirmed by tissue evidence in 200; the other 14 patients had either no evidence of prostatic carcinoma on surgical biopsy (needle biopsy/transurethral resection/suprapubic prostatectomy) or had no surgical biopsy. Eight of the 14 patients developed clinical evidence of carcinoma, 1 died of urinary bladder carcinoma and 1 was lost to follow-up. In the remaining four patients, there is still no evidence of prostatic carcinoma after about one-and-one-half years of follow-up. These results indicate that (1) specialized training is required in order to obtain adequate smears by prostatic FNA; (2) prostatic FNA is not a good screening technique for detecting stage A1 prostatic carcinoma; and (3) a positive diagnosis by prostatic FNA, even when not confirmed by tissue biopsy, is still an indication of disease.     

The Role of Core-Needle Biopsy for Thyroid Nodules with Initially Nondiagnostic Fine-Needle Aspiration Results: A Systematic Review and Meta-Analysis

Objective: This study evaluated the prevalence of nondiagnostic results, diagnostic performance, and complications of core-needle biopsy (CNB) compared with repeat fine-needle aspiration (FNA) for thyroid nodules with previous nondiagnostic FNA findings.Methods: The Ovid-MEDLINE and EMBASE databases were thoroughly searched for studies evaluating CNB or repeat FNA for thyroid nodules with initially nondiagnostic FNA results. Pooled proportions of nondiagnostic results of CNB and repeat FNA were calculated. A meta-analysis was performed to evaluate the diagnostic accuracy of CNB and repeat FNA for a diagnosis of malignancy using a bivariate random-effects model. Complication rates were also evaluated.Results: A review of 52 articles identified 4 eligible articles, involving 1,028 patients with 1,028 thyroid nodules, which were included in the meta-analysis. CNB demonstrated significantly lower rates of nondiagnostic results (6.4%) than repeat FNA (36.5%) (P<.0001). In the 3 studies that analyzed the diagnostic accuracy of CNB in diagnosing malignancy, CNB demonstrated significantly higher summary estimates of sensitivity (89.8%) than repeat FNA (60.6%) (P = .022), but summary specificity did not differ between CNB (99.2%) and repeat FNA (99.0%) (P = .576). None of the patients who underwent CNB or repeat FNA experienced any major complications.Conclusion: CNB demonstrates lower rates of nondiagnostic results and higher diagnostic accuracy than repeat FNA. CNB, rather than repeat FNA, can be utilized to diagnose thyroid nodules previously nondiagnostic on FNA.Abbreviations:CI = confidence intervalCNB = core-needle biopsyFNA = fine-needle aspirationSROC = summary receiver operating characteristicUS = ultrasound     

Galectin-3 is not useful in thyroid FNA

INTRODUCTION: Previous studies have suggested that galectin-3 immunohistochemistry may be useful in the fine needle aspiration (FNA) diagnosis of thyroid carcinoma as it has been reported to selectively stain carcinomas and not adenomas or goitres. METHODS: Fifty-one patients were included in a prospective study of galectin-3 in thyroid FNA; 88.2% were female and 11.8% male, mean age 53 years, range 25-87 years. Cell blocks were prepared and stained for galectin-3 if any cells were present in needle washings from the respective FNAs. RESULTS: Twelve of 51 (23.5%) of cell blocks contained epithelial cells. One benign and one inadequate FNA were negative for galectin-3 staining. One of five non-diagnostic FNA cases, a papillary carcinoma on final histology showed positive staining. Four follicular neoplasm/suspicious of carcinoma cases showed negative staining. One malignant FNA case, a papillary carcinoma showed positive staining with galectin-3 but three further carcinomas, two papillary and one follicular were galectin-3 negative. CONCLUSION: Galectin-3 immunohistochemistry does not appear to be a useful adjunct to diagnosis in thyroid FNA as it does not reliably distinguish malignant and benign lesions. Many thyroid aspirates are of low cellularity and are not suitable for cell block immunohistochemistry.     

Fine needle aspiration (FNA) of testicular germ cell tumours; a 10-year experience in a community hospital

garcía-solano j., sánchez-sánchez c., montalbán-romero s., sola-pérez j. and pérez-guillermo m. (1998) Cytopathology 9, 248–262
Fine needle aspiration (FNA) of testicular germ cell tumours; a 10-year experience in a community hospital
A retrospective reappraisal is made of the smears of 29 testicular germ cell tumours (TGCT) studied by FNA in which both orchiectomy specimens and histologic diagnoses were available. In 22 cases (75.86%) the yield was sufficient and contained cells suitable for cytologic diagnosis; in these 22 cases a diagnosis of malignancy was reached. In four cases (13.79%) the yield was sparse and diagnostic cells were partially obscured by haemorrhage and necrosis; these cases were categorized as suspicious of malignancy. In three cases (10.34%) the yield was not suitable for cytologic evaluation because haemorrhage and necrosis hampered evaluation of diagnostic cells. The cytologic findings that enable a reliable diagnosis of TGCT are described and those cytologic features that may lead the less experienced cytopathologist into an erroneous diagnosis are discussed. Pure TGCT can be confidently diagnosed with FNA and mixed TGCT can be successfully diagnosed, although it is difficult to recognize every cytologic subtype observed in the histologic sections. Despite the advantages of FNA for the prompt diagnosis of TGCT, FNA can not fully replace the histologic diagnosis and should rather be considered as a helpful tool in the work-up of testicular tumours.     

Necrosis in thyroid nodules after fine needle aspiration biopsy. Report of two cases

Two cases of infarction of thyroid neoplasms following fine needle aspiration (FNA) biopsy are reported. Histologic study of a 2.5 x 2.5 cm nodule excised 18 days after FNA had diagnosed a Hürthle-cell neoplasm showed mainly necrotic debris and granulation tissue. While FNA made the diagnosis of a papillary carcinoma in the second case, which had had an FNA biopsy of the same nodule six years earlier, most of the nodule was fibrotic and necrotic. These two cases demonstrate the potential problems in such cases: (1) post-FNA infarction may obscure the nature of a cytologically diagnosed neoplasm, making histologic confirmation difficult, and (2) FNA of an infarcted nodule may have difficulties in obtaining diagnostic material, potentially resulting in a false-negative diagnosis. Review of the literature on thyroid infarction shows it to be a rare event, with most reported cases occurring after FNA biopsy of a neoplasm. The finding of necrosis and fibrosis in an aspirate or surgical specimen should thus suggest the presence of a neoplasm.     

Expansion of tumor-T cell pairs from fine needle aspirates of melanoma metastases

Lymphocytes expanded from excised specimens can be used to characterize intratumoral T cell responses. These analyses, however, are limited to one time point in the natural history of the removed tumor. The expansion of autologous tumor cells and tumor-infiltrating lymphocytes (TIL) from fine needle aspirates (FNA) of tumors potentially allows a dynamic evaluation of T cell responses within the same lesion at moments relevant to the disease course or response to therapy. Fourteen TIL cultures and 8 tumor cell lines were generated from 18 FNA (12 patients). Five of six TIL that could be tested against autologous tumor demonstrated specific reactivity. Two additional TIL for which no autologous tumor was available demonstrated recognition of HLA-matched melanoma cell lines. Serial FNA of the same lesions were performed in five HLA-A*0201 patients vaccinated with the emulsified melanoma Ag (MA) epitopes: MART-1:27-35; tyrosinase:368-376(370D); gp100:280-288(288V); and gp100:209-217 (210M). FNA material was separately cultured for a short time in IL-2 (300 IU/ml) after stimulation with irradiated autologous PBMC pulsed with each peptide or FluM1:58-66 (1 micromol/ml). No peptide-specific TIL could be expanded from prevaccination FNA. However, after vaccination, TIL specific for gp100:280(g280), gp100:209 (g209), and MART-1:27-35 (MART-1)-related epitopes were identified in three, three, and two patients, respectively. No Flu reactivity could be elicited in TIL, whereas it was consistently present in parallel PBMC cultures. This excluded PBMC contamination of the FNA material. This analysis suggests the feasibility of TIL expansion from minimal FNA material and localization of vaccine-specific T cells at the tumor site.