We found that the use of p53 and IMP3 immunostains can be helpful in distinguishing reactive atypia from dysplasia, when dealing with these challenging case scenarios in cholecystectomy specimens. In particular, we found that negative IMP3 stain in conjunction with wild-type p53 staining would support a diagnosis of reactive atypia over true dysplasia.
GBC is considered to develop from a metaplasia-dysplasia-carcinoma sequence, making the diagnosis of dysplasia very important [5]. RA is a benign entity occurring in inflamed epithelium, but as dysplasia is frequently associated with chronic inflammation, dysplasia is often difficult to distinguish histologically from RA. Complete resection of gallbladder with dysplasia along with negative cystic duct resection margins is sufficient prophylactic treatment for GBC in these cases [11]. However, in addition to the possible progression of dysplasia to GBC, Rais et al. found that in cases with incidental gallbladder dysplasia, 18.9% of patients had an associated pancreatobiliary carcinoma [11]. Given the hypothesis of multifocal neoplastic potential in the pancreatobiliary tree, patients with dysplasia in the gallbladder would benefit from monitoring for development of other neoplasia of the biliary tract.
Wild-type p53 has functions in tumor suppression through cell cycle arrest, apoptosis, transcription, DNA repair, etc. It has a central role in preventing malignant progression, thus accumulation of genetic damage increases tendency for carcinogenesis [12]. It has previously been thought that p53 expression marked a late event in gallbladder carcinogenesis [13]. In a study by Legan et al., all NL and LGD gallbladder cases showed wild-type p53, whereas 31.2% of their HGD cases were p53 aberrant. They postulated that any p53 expression in LGD could be due to failure of p53 degradation, leading to accumulation of wild-type protein and thus pushing the cell into cycle arrest and apoptosis. These cells would not proliferate as malignant clones [14].
However, loss of a chromosomal region in the TP53 gene has been found in normal-appearing and dysplastic gallbladder epithelium, suggesting that TP53 abnormalities precede protein overexpression in the pathogenesis of GBC [12, 15]. P53 mutations have also been linked with dysplastic epithelium in patients with gallstone disease, indicating a chronic inflammatory cascade [16]. The results from a study by Wee et al. further reinforces this as they found 50% of dysplastic gallbladder tissue showed p53 aberrancy, with 28% expressing strongly (> 50%) [17]. In fact, a case report of a patient who had confirmed complete GBC resection post-cholecystectomy was found to have over-expression of mutant p53 in the normal surrounding epithelium of the cystic duct. This patient developed bile duct cancer only 2.5 years later [18]. Our study found 10% of RA, 50% of dysplasia, and 100% of IAC cases show mutated p53 on IHC. This supports the role that aberrant p53 has in gallbladder cancer pathogenesis as areas of strong staining could imply protein mutation has already occurred. In fact, it has been hypothesized that dysplasia with mutant p53 evolve into more aggressive tumor types with higher grade [19].
IMP3 expression in gallbladder carcinomas and dysplasia has shown contrasting findings. Kim et al. found negative IMP3 expression in dysplasia and chronic cholecystitis specimens while 87.7% of GBC expressed IMP3 immunoreactivity, concluding that IMP3 is a useful diagnostic marker for GBC. They also noted that strong IMP3 expression was associated with higher histological grade, advanced age, lymphatic invasion, and worse overall survival [20]. Some other studies have also supported similar findings of IMP3 predicting poor prognosis and the presence of invasion in biliary tract carcinomas [21, 22].
On the other hand, Riener et al. claimed IMP3 to be useful for HGD diagnosis in the extrahepatic biliary tract. In their study, all HGD demonstrated strong IMP3 staining, while all NL and LGD had weak or no IMP3 reactivity. This resulted in a sensitivity and specificity of 1 for IMP3 use in HGD in the extrahepatic biliary tract. They also found differences in IMP3 expressions in different types of cancers. GBC expressed IMP3 most strongly at 81.6%, whereas intra- and extra-hepatic cholangiocarcinoma showed IMP3 positivity rates of 36.8% and 50%, respectively [8]. While our study showed a similar IAC IMP3 expression of 80%, some RA cases in our study had positive IMP3 expression. We found that IMP3 is valuable in differentiating RA from LGD and HGD. These findings may be related to a difference in etiology and molecular alterations seen in gallbladder lesions compared to those from the rest of the biliary tract. There could be an earlier expression of IMP3 in the gallbladder compared to the extrahepatic bile ducts, or the IMP3 positive gallbladder cases, like those with mutant p53, are likely to become more aggressive later in their courses.
The utility of S100P in the gallbladder remains controversial. During embryonic development, S100P is expressed in several tissues, including the epithelium of the gallbladder [23]. In adults, S100P has been widely expressed in both normal and malignant tissues [24]. One study demonstrated negative S100P staining in NL gallbladder tissue and positive staining in 50% of chronic cholecystitis cases. They also noted positive staining of background stroma, inflammatory, and endothelial cells [25]. Another study found that S100P expression was significantly higher in GBC (61.7%) than in benign tissues [26]. However, the analysis from this present study indicates that S100P does not aid in gallbladder disease differentiation as the stain showed pan-positivity from NL to IAC tissue.
It is also worth mentioning that whether S100P should be used as a prognostic marker in GBC remains inconclusive. While one study found S100P to be associated with more advanced GBC disease and poor survival, their study was limited by the lack of a control group [27]. A meta-analysis conducted by Liu et al. concluded that S100P was not a helpful predictor of overall survival in patients with gallbladder cancer [28].
Due to the rare nature of gallbladder dysplasia and carcinomas, our study was limited by study size, especially the number of HGD specimens. A larger cohort with ample cases across all categories may provide stronger significance. Inevitably, interobserver variability in diagnosis among pathologists should also be noted, though we attempted to minimize this by requiring a consensus among the 3 GI subspecialized pathologists in this study. We also note that the cutoff ranges for a positive result vary from study to study, making direct comparisons difficult. A study in which the patients are followed longitudinally to assess for prognosis and development of neoplasia or worsening disease could provide greater insight into the role these proteins have on disease prediction and progression.
In summary, we conclude that IMP3 and p53 IHC stains showed statistically significant differences when differentiating RA from LGD and HGD, while S100P demonstrated no significant benefit. Morphology on H&E staining remains the standard for RA and dysplasia diagnosis; however, in cases that are difficult to distinguish, immunopositivity for IMP3 or p53 aberrant expression may help favor a diagnosis of LGD over RA.
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