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FISHing: New Methods to Improve the Diagnostic Sensitivity of Fine Needle Aspiration Cytology

      See “Comparison of methods to detect neoplasia in patients undergoing endoscopic ultrasound-guided fine-needle aspiration,” by Levy MJ, Oberg TN, Campion MB, et al, on page 1112.
      Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) has proven to be a major advance in endoscopy through its ability to obtain cytologic material from lymph nodes, mural lesions, and extra-gastrointestinal masses. Although EUS has become remarkably effective at imaging and aspirating these lesions, the cytologic sensitivity has remained rather modest. In the initial description of EUS FNA, Wiersema et al
      • Wiersema M.J.
      • Hawes R.H.
      • Tao L.C.
      • et al.
      Endoscopic ultrasonography as an adjunct to fine needle aspiration cytology of the upper and lower gastrointestinal tract.
      determined that the sensitivity rates for lymph nodes, masses, and wall lesions was 92%, 88%, and 61%.
      Over the past 15 years, there has been a major effort to improve the results of EUS FNA cytology by providing more and higher quality cytologic material. Initially, endoscopists were forced to obtain a large amount of aspirated material by performing a large number of needle passes. Pancreatic masses were found to be the most difficult lesions from which diagnostic material could be obtained.
      • Gress F.
      • Gottlieb K.
      • Sherman S.
      • et al.
      Endoscopic ultrasonography-guided fine-needle aspiration biopsy of suspected pancreatic cancer.
      Initially, large-gauge needles were thought to provide a higher rate of diagnostic specimens from the pancreas; however, over time it has become apparent that the size of the needle may not be a strong determinant of the diagnostic quality of cytology.
      • Lee J.H.
      • Stewart J.
      • Ross W.A.
      • et al.
      Blinded prospective comparison of the performance of 22-gauge and 25-gauge needles in endoscopic ultrasound-guided fine needle aspiration of the pancreas and peri-pancreatic lesions.
      Different needle designs have also been investigated in hopes of improving the quality of malignant tissue. Although Tru-cut needles have been very good for securing histologic material from benign pancreatic diseases such as autoimmune pancreatitis, these cutting needles do not provide a higher diagnostic rate for pancreatic cancer.
      • Sakamoto H.
      • Kitano M.
      • Komaki T.
      • et al.
      Prospective comparative study of the EUS guided 25-gauge FNA needle with the 19-gauge Trucut needle and 22-gauge FNA needle in patients with solid pancreatic masses.
      A number of different aspiration techniques have also been attempted. The degree of suction does not seem to be terribly important.
      • Larghi A.
      • Noffsinger A.
      • Dye C.E.
      • et al.
      EUS-guided fine needle tissue acquisition by using high negative pressure suction for the evaluation of solid masses: a pilot study.
      The use of stylet does not improve the diagnostic rate of cytology.
      • Wani S.
      • Gupta N.
      • Gaddam S.
      • et al.
      A comparative study of endoscopic ultrasound guided fine needle aspiration with and without a stylet.
      It seems that the presence of blood is a very important factor that reduces the ability of the cytologist to provide a diagnostic interpretation.
      • Lee J.K.
      • Choi E.R.
      • Jang T.H.
      • et al.
      A prospective comparison of liquid-based cytology and traditional smear cytology in pancreatic endoscopic ultrasound-guided fine needle aspiration.
      In fact, the only consistent technique to improve the diagnostic rate has been the presence of an on-site cytologyist.
      • Iglesias-Garcia J.
      • Dominguez-Munoz J.E.
      • Abdulkader I.
      • et al.
      Influence of on-site cytopathology evaluation on the diagnostic accuracy of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of solid pancreatic masses.
      Because the diagnostic sensitivity of aspirated tissue is highly dependent on the interpretation of cytologic findings, a subjective finding, it might be useful to be able to provide an objective finding in the aspirated tissue to provide a diagnosis. This approach has been very successful in the cytologic specimens aspirated from lymph nodes of patients with lung cancer.
      • Wallace M.B.
      • Block M.
      • Hoffman B.J.
      • et al.
      Detection of telomerase expression in mediastinal lymph nodes of patients with lung cancer.
      Telomerase expression has been detected with the use of human telomerase reverse transcriptase. Tissue staining for p53 and ki67 has been used for pancreatic FNA cytology specimens.
      • Salek C.
      • Benesova L.
      • Zavoral M.
      • et al.
      Evaluation of clinical relevance of examining K-ras, p16 and p53 mutations along with allelic losses at 9p and 18q in EUS-guided fine needle aspiration samples of patients with chronic pancreatitis and pancreatic cancer.
      It has been well established that the loss of chromosome fragments is a common finding in malignant tissue and this can be detected with techniques such as loss of heterozygosity analysis.
      • Salek C.
      • Benesova L.
      • Zavoral M.
      • et al.
      Evaluation of clinical relevance of examining K-ras, p16 and p53 mutations along with allelic losses at 9p and 18q in EUS-guided fine needle aspiration samples of patients with chronic pancreatitis and pancreatic cancer.
      Loss of chromosomal alleles linked to known tumor suppressor genes, high amplitude k-ras mutation, and high DNA amount (presumably reflecting high mitosis rate) have all been shown to complement traditional imaging and cytologic analysis.
      • Khalid A.
      • Zahid M.
      • Finkelstein S.D.
      • et al.
      Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study.
      These findings have been exploited through the use of relatively new cytologic technique, fluorescence in situ hybridization (FISH).
      • Reicher S.
      • Boyar F.Z.
      • Albitar M.
      • et al.
      Fluorescence in situ hybridization and K-ras analyses improve diagnostic yield of endoscopic ultrasound-guided fine-needle aspiration of solid pancreatic masses.
      FISH, a cytogenetic technique, is used to detect and localize the presence or absence of specific DNA sequences on chromosomes (Figure 1). FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes. In the study by Levy et al,
      • Levy M.J.
      • Oberg T.N.
      • Campion M.B.
      • et al.
      Comparison of methods to detect neoplasia in patients undergoing endoscopic ultrasound- guided fine-needle aspiration.
      in this issue of Gastroenterology, a probe developed for genital–urinary malignancy was used to detect evidence of chromosomal abnormalities in gastrointestinal cancer. The diagnostic rate of FISH was found to be very similar to conventional cytology (77%). However, when FISH was added to conventional cytology, the diagnostic rate increased to 86%.
      Figure thumbnail gr1
      Figure 1The figure demonstrates the conceptual framework for FISH methods. The overall goal is to identify chromosomal additions or deletions by fluorescence labeling if key sites on chromosomes linked to known cancer-associated genes.
      The role of FISH analysis awaits further work on its clinical utility. Obviously, FISH analysis should not be performed routinely on all specimens, particularly diagnostic specimens. The application of FISH analysis should focus on nondiagnostic specimens, looking for evidence of undetected malignancy. Eighty percent of specimens with cytologic atypia have a positive FISH or kRAS mutation.
      • Reicher S.
      • Boyar F.Z.
      • Albitar M.
      • et al.
      Fluorescence in situ hybridization and K-ras analyses improve diagnostic yield of endoscopic ultrasound-guided fine-needle aspiration of solid pancreatic masses.
      Levy has reported very similar results in a subset of patients with a non-diagnostic cytology. Of 69 patients suspicious with cytology, 13 were found to have malignancy by FISH analysis.
      • Kubiliun N.
      • Ribeiro A.
      • Fan Y.S.
      • et al.
      EUS-FNA with rescue fluorescence in situ hybridization for the diagnosis of pancreatic carcinoma in patients with inconclusive on-site cytopathology results.
      FISH and other similar methods are limited by the number of alleles that can be evaluated, and the inherent bias toward assessment of known oncogenes and tumor suppressor genes. Recent advances in high-throughput, rapid sequencing of whole genomes or exons, with the ability to detect single base-pair mutations or fusion genes, offers even greater potential to discover diagnostic, prognostic, and predictive biomarkers, as well as much higher specificity.
      • Asmann Y.W.
      • Wallace M.B.
      • Thompson E.A.
      Transcriptome profiling using next-generation sequencing.
      It seems that the application of molecular testing to specimens obtained with EUS FNA will improve the diagnostic sensitivity of cytology in the detection of malignancy. Ultimately, our goals should be to personalize the approach to neoplasms, identifying those individuals at very low risk who need no therapy and identifying targeted treatment options for those at higher risk of malignancy.

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