To the Editor:

We read with great interest the article by Bangarulingam et al. regarding the long-term outcomes of positive fluorescence in situ hybridization (FISH) in patients with primary sclerosing cholangitis (PSC).1 We applaud the authors for applying bile duct cytology and FISH to a large cohort of patients with PSC to better characterize the long-term outcomes. The authors used Vysis UroVysion, a commercially available kit that was approved by the U.S. Food and Drug Administration in 2005 for use in the initial diagnosis of bladder cancer in patients with hematuria.2 This probe set has since been applied to detect chromosomal abnormalities in various body sites including the detection of malignancies in biliary strictures.1, 3-7 The UroVysion kit allows for the simultaneous testing of numeric aberrations, or aneusomy, of chromosome 3 (CEP3), chromosome 7 (CEP7), and chromosome 17 (CEP17), as well as band 9p21 (P16/CDKN2A) deletions.

Unfortunately, the authors provide no information on the results of CEP17 and p16 abnormalities in their cohort. We view the omission of the CEP17 and p16 results as a potential lost opportunity. In histology specimens, p16 inactivation has been shown to be common in PSC-associated cholangiocarcinoma (CCA) with 90% showing the loss of one allele which correlated with the loss of p16 expression in 57% of CCAs.8 Functional point mutations in the p16 promoter likely contribute to the initiation and progression of PSC-associated CCA.9 Using FISH, it was reported that four of six PSC-associated CCAs had CEP3, CEP7, and CEP17 aneusomy.5 The two CCAs that did not have aneusomy had p16 deletions.5 In addition, 64% of CCAs had CEP17 aneusomy, compared to 82% and 77% with aneusomy of CEP3 and CEP7, respectively.5 It appears that CEP17 aneusomy and p16 deletions may be more common in PSC-associated CCA than the authors report.

Since 2008, our liver program has adopted the use of FISH in addition to cytology in the diagnosis of indeterminate strictures and PSC-associated dominant strictures (n = 56). In our initial series, 12 tissue-proven CCAs were identified, of which 9 had nondiagnostic cytology.10 As reported previously, CEP3 and CEP7 aneusomy were most commonly seen in CCA (7 of 12 CCAs). Among CCA cases with positive FISH and negative cytology, we found that CEP17 aneusomy was present in 75% and p16 deletions were seen in 50%. Among the cases that had a p16 deletion (homozygous or heterozygous), nearly half of the cases (5 of 9) had no other chromosomal changes. Based on our experience and previously published data, we believe that the inclusion of CEP17 and p16 status may have significant additional diagnostic importance.

After reviewing their published data, we agree with the author's conclusion that FISH is inadequate to be used as a CCA screening modality in unselected patients with PSC, but may have a role in patients with a clinical or laboratory suspicion for PSC-associated dominant strictures. However, we question if their conclusion would have changed with the inclusion of CEP17 aneusomy and/or p16 deletions. Because these results are currently available to the authors for all patients, we ask that they reconsider the exclusion of this data from interpretation.


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  • 1
    Bangarulingam SY, Bjornsson E, Enders F, Barr Fritcher EG, Gores G, Halling KC, et al. Long-term outcomes of positive fluorescence in situ hybridization tests in primary sclerosing cholangitis. HEPATOLOGY 2010; 51: 174-180.
  • 2
    Halling KC, King W, Sokolova IA, Meyer RG, Burkhardt HM, Halling AC, et al. A comparison of cytology and fluorescence in situ hybridization for the detection of urothelial carcinoma. J Urol 2000; 164: 1768-1775.
  • 3
    Moreno-Luna LE, Kipp B, Halling KC, Sebo TJ, Kremers WK, Roberts LR, et al. Advanced cytologic techniques for the detection of malignant pancreatobiliary strictures. Gastroenterology 2006; 131: 1064-1072.
  • 4
    Kipp BR, Stadheim LM, Halling SA, Pochron NL, Harmsen S, Nagorney DM, et al. A comparison of routine cytology and fluorescence in situ hybridization for the detection of malignant bile duct strictures. Am J Gastroenterol 2004; 99: 1675-1681.
  • 5
    DeHaan RD, Kipp BR, Smyrk TC, Abraham SC, Roberts LR, Halling KC. An assessment of chromosomal alterations detected by fluorescence in situ hybridization and p16 expression in sporadic and primary sclerosing cholangitis-associated cholangiocarcinomas. Hum Pathol 2007; 38: 491-499.
  • 6
    Barr Fritcher EG, Kipp BR, Slezak JM, Moreno-Luna LE, Gores GJ, Levy MJ, et al. Correlating routine cytology, quantitative nuclear morphometry by digital image analysis, and genetic alterations by fluorescence in situ hybridization to assess the sensitivity of cytology for detecting pancreatobiliary tract malignancy. Am J Clin Pathol 2007; 128: 272-279.
  • 7
    Levy MJ, Baron TH, Clayton AC, Enders FB, Gostout CJ, Halling KC, et al. Prospective evaluation of advanced molecular markers and imaging techniques in patients with indeterminate bile duct strictures. Am J Gastroenterol 2008; 103: 1263-1273.
  • 8
    Ahrendt SA, Eisenberger CF, Yip L, Rashid A, Chow JT, Pitt HA, et al. Chromosome 9p21 loss and p16 inactivation in primary sclerosing cholangitis-associated cholangiocarcinoma. J Surg Res 1999; 84: 88-93.
  • 9
    Taniai M, Higuchi H, Burgart LJ, Gores GJ. p16INK4a promoter mutations are frequent in primary sclerosing cholangitis (PSC) and PSC-associated cholangiocarcinoma. Gastroenterology 2002; 123: 1090-1098.
  • 10
    Glick M GT, Iqbal S, Nandula S, Kang JU, Murty V, Stevens P, et al. Fluorescent in situ hybridization in the diagnosis of indeterminate biliary strictures [Abstract]. Am J Gastroenterol 2009; 104( Suppl. 3): 177.

Lance L. Stein M.D.*, Tamas A. Gonda M.D.†, Peter D. Stevens†, Robert S. Brown Jr. M.D., M.P.H.*, * Department of Medicine, Center for Liver Disease and Transplantation, Columbia University College of Physicians and Surgeons, New York, NY, † Department of Medicine, Division of Gastroenterology, Columbia University College of Physicians and Surgeons, New York, NY.