We would like to thank Dr. L. Stein and colleagues for the comments on our article regarding long-term outcomes of positive fluorescence in situ hybridization (FISH) in patients with primary sclerosing cholangitis (PSC).1
With regard to the major concern about not providing information on results for CEP17 and P16, we were aware that the P16 (at 9p21) and P53 (on 17p) genes are frequently inactivated through gene deletion, point mutation, or promoter hypermethylation in cholangiocarcinoma (CCA). The only one of these abnormalities detectable with FISH is gene deletion. It is important to understand that the results we showed were for a clinically developed and validated test and not a research study. The reason data was not provided for P16 is that when the assay was developed and validated for clinical use, we found it difficult to use the 9p21 (P16) probe for discerning cancer. One of the reasons was that the 9p21 probe is yellow and bile autofluoresces yellow, so it was difficult to see the 9p21 probe and to be comfortable with the number of copies of 9p21 in a cell. We no longer use bile aspirates, so that is not really an issue anymore (i.e., there is not a problem with yellow autofluorescence in bile duct brushings).
The more important reason we decided not to use 9p21 when this was clinically implemented in 2003 is that it would have made analyzing the specimens more complicated and laborious. We wanted an assay that could be done in a reasonable amount of time. The reason that it would create more work is that 9p21 is directed to the P16 tumor suppressor gene and the change that one would be looking for is deletion of the 9p21 probe (one or zero copies). It should be understood that a low but appreciable fraction of normal cells show what looks like 9p21 deletion. For example, an average of about 10% of normal cells might be expected to show only a single copy of 9p21. This is not due to real deletion but overlap of the two probe signals such that it appears to be a single copy. Because of this, to feel comfortable calling a case positive for 9p21 loss without generating false positive calls would require having a cutoff of well over 10% of the cells showing hemizygous 9p21 loss. To capture this information would require formal enumeration of 50 or 100 cells and increase the amount of time required to clinically interpret the assay. As indicated before, we wanted a clinical assay that was not too laborious. However, we recognized that we might have some false negative results because we might miss cases that showed only 9p21 loss without any chromosomal gains. So, the bottom line for 9p21 (P16) is that Stein et al. are correct that we probably failed to detect some cases of CCA due to not assessing for this. Once again, it is important to realize that this was a clinical study and not a research study. We could always go back and try to capture that data but it would not reflect the clinical test that was used.
With regard to CEP17, aneusomy can refer to gain or loss of a probe. Stein et al. do not state which type of CEP17 aneusomy they were interested in knowing more about. If they are referring to loss of CEP17, then the reason we did not capture that information is for the same reasons listed above, i.e., that it would require a more laborious enumeration due to the low-level artifactual CEP17 loss that exists in normal cells. If they are referring to gain of CEP17, then even though we did not specifically capture information on CEP17 gain, cells with CEP17 gain would have been categorized as trisomic (if only CEP17 was gained, which is very rare in our patient population) or polysomic (if CEP17 was gained along with gain of one or more of the other probes), and thus that information was captured within those diagnostic categories. Incidentally, we know that cases with P53 loss (due to partial or complete loss of chromosome 17) tend to show chromosomal instability (gains) which manifests as polysomy by FISH. For this additional reason, CEP17 (P53) loss is unlikely to add any additional sensitivity beyond that obtained with polysomy.
We are currently working to develop a FISH enumeration procedure that will allow us to use 9p21 loss as a criterion for positivity in the clinical assay that we use. This should allow us to increase the sensitivity of the assay.