Material and Method: With the participation of 22 institutions around Turkey that were included in the Hepato-Pancreato-Biliary Study Group, 89,324 cholecystectomy specimens sampled from 2003 to 2016 were retrospectively evaluated. The numbers of adenocarcinomas, dysplasias, intracholecystic neoplasms/adenomas, intestinal metaplasias and reactive atypia were identified with the review of pathology reports and the regional and countrywide incidence rates were presented in percentages.

Results: Epithelial changes/lesions were reported in 6% of cholecystectomy materials. Of these epithelial lesions, 7% were reported as adenocarcinoma, 0.9% as high-grade dysplasia, 4% as low-grade dysplasia, 7.8% as reactive/regenerative atypia, 1.7% as neoplastic polyp, and 15.6% as intestinal metaplasia. The remaining lesions (63%) primarily included non-neoplastic polypoids/hyperplastic lesions and antral/pyloric metaplasia. There were also differences between pathology laboratories.

Conclusion: The major causes of the difference in reporting these epithelial changes/lesions and neoplasms include the differences related to the institute's oncological surgery frequency, sampling protocols, geographical dissimilarities, and differences in the diagnoses/interpretations of the pathologists. It seems that the diagnosis may change if new sections are taken from the specimen when any epithelial abnormality is seen during microscopic examination of the cholecystectomy materials.

Epithelial changes/lesions and neoplasms of the gallbladder are rarely encountered lesions^1,2. However, as cholecystectomy is a relatively common surgical procedure, epithelial lesions of the gallbladder, as rare as they are, may be confronted by surgical pathologists. Among the epithelial changes/lesions of the gallbladder, one can count metaplastic changes/lesions (antral/pyloric metaplasia, intestinal metaplasia), benign epithelial neoplasms (adenomas/cystadenomas), biliary intraepithelial neoplasias (BilIN) (dysplasia/carcinoma in situ), and invasive carcinomas². The protocols to be followed in sampling and microscopic examination of the gallbladder are still of paramount importance and continue to be an issue of debate as high-grade dysplasia and even invasive carcinoma cannot be macroscopically identified to a large extent³.

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Figure 1: Mass forming invasive adenocarcinoma of gallbladder.

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Figure 2: Invasive adenocarcinoma of gallbladder (H&E; x100).

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Figure 3: High-grade dysplasia in gallbladder epithelium (H&E; x400).

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Figure 4: Low-grade dysplasia in gallbladder epithelium (H&E; x400).

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Figure 5: Reactive atypia in gallbladder epithelium nearby intestinal metaplasia (H&E; x400).

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Figure 6: Intestinal metaplasia in gallbladder epithelium (H&E; x400).

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Figure 7: Antral metaplasia in gallbladder epithelium (H&E; x200).

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Figure 8: The percentages of epithelial lesions detected in cholecystectomy specimens.

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Table I: The percentages of epithelial changes/lesions detected in cholecystectomy specimens

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Table II: The percentages of accompanying epithelial lesions in the presence of intestinal metaplasia

The geographical distribution of certain important lesions is presented in Table III. Because results from the Black Sea Region were obtained from only one centre, specimens from that region are relatively limited in number. According to the available data, the highest rates of invasive adenocarcinoma was found in the Aegean Region while the highest rate of high-grade dysplasia was in yjr Mediterranean Region, the highest rate of reactive atypia and intestinal metaplasia was in the Black Sea Region, and the highest rate of biliary adenoma was in the Central Anatolia Region.

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Table III: Distribution of certain major epithelial lesions by geographical regions

Gallbladder carcinomas are usually incidentally detected in cholecystectomy specimens taken out due to cholestasis and display themselves as carcinoma in situ or carcinoma with superficial invasion². In our study, the rate of primary invasive adenocarcinoma in all cholecystectomy specimens was found to be 0.4% and this rate is reported in the literature to be between 0.15-2.3%^4-10. In addition, the world-wide incidence, mortality, and prevalence rate for 5 years of gallbladder cancer is reported, respectively, to be 1.3%, 1.7%, and 06% in the GLOBOCAN Project that establishes up-to-date estimates at the national level about the incidence, mortality, and prevalence for major cancers in many countries¹¹. In our study, the prevalence rate for 5 years for gallbladder cancer was found to be 0.5% for Turkey and this figure is in accordance with the data provided by GLOBOCAN.

Gallbladder dysplasia is not a frequent condition and its incidence is reported to be 3.3% ¹². However, there are studies in the literature that claim this rate can go up to 25% ^13-16. The rate of dysplasia detected in all cholecystectomy specimens in our study, on the other hand, was 0.25%. The number of studies in the literature that focuses on this issue is not adequate but the rate is reported to be between 0.7-3.3% in all studies available ^5-7,12. The fact that the rate found in our study is well below the ones reported in the literature suggests that either these pre-neoplastic epithelial lesions could not be sampled or not recognized enough or low-grade dysplasia was misinterpreted as regenerative atypia. It is reported in different series that the incidence rate of intestinal metaplasia is 10-30% among all cholecystectomy specimens ². A striking result in this respect was found in a study conducted in the population of Chile, which is known to have a relatively higher rate of gallbladder cancer. The rate of intestinal metaplasia detected in cholecystectomy specimens was reported in this study to be 58.1% ¹⁷. In our study, the relevant result was 0.9%. This figure was again below the ones reported in the literature and it was thought that this results from the discrepancies in routine reporting of intestinal metaplasia in pathology reports by pathologists and/or institutions and the non-uniformity in macroscopic sampling.

For macroscopic examination and sampling of cholecystectomy specimens, it was recommended in the handbook of macroscopic examination ¹⁸ prepared by the HPBSG of the Federation of Turkish Pathology Societies in 2013 that all cholecystectomy specimens should be handled according to the following: (i) The specimen should be measured in three dimensions, opened on the anti-hepatic side and the bile drained off. It should be noted whether the gallbladder has already been opened and the container in which the specimen has been sent should be investigated for lesions that may have come off. The specimen should not be opened in the sink under running water. (ii) The number, size, shape and colour of the gallstones should be described. (iii) The mucosa should be examined thoroughly and its appearance, yellow mottlings, and whether it has an ulcer or a mass lesion be specified. (iv) The thickness of the gallbladder wall should be documented based on the thickest part of the specimen. (v) The neck (isthmic) lymph node (so called “Cattle ganglion”) should meticulously be searched for and sampled completely after being cut into two. (iv) The surgical margin of the cystic duct and the hepatic aspect of the gallbladder should be marked with ink and the samples from the peritonealized surface of the organ should be noted, and also painted with ink. This handbook also includes recommendations on the sampling method and the number of slices to be sampled in cholecystectomies performed due to tumour-associated or non-tumour-associated reasons or due to the existence of tumour suspicion on macroscopic examination.

It is also noticed that various sampling methods are available for the sampling of cholecystectomy specimens in the reference books of pathology that are in current international use. Among these are: (i) especially for the specimens with a prediagnosis of carcinoma, draining off the bile with the help of an injector, injecting formalin in the bladder, leaving the specimen to be fixed in formalin overnight and then opening and sampling; for other specimens, sampling three pieces each from the fundus, body, and the neck to represent the whole wall of the gallbladder and sampling cystic duct and any lymph node in case of any macroscopic abnormality ¹⁹; (ii) sampling by multiple transverse sections after rolling the emptied organ to examine the whole mucosa on a single slide, especially for the specimens with suspicion of carcinoma in situ or dysplasia ¹²; (iii) sampling by circumferential transverse sectioning of the proximal cystic duct margin and by one slice divided into appropriate pieces obtained from a whole cut transverse section of the cholecystectomy specimens without any macroscopic abnormality ²⁰; (iv) sampling of the fundus and neck of the gallbladder each cut in the transverse plane and the margin of cystic duct with fine transverse sectioning ²¹.

There are also several studies in our country on the prevalence of epithelial lesions in the gallbladder mucosa. As for dysplasia and intestinal metaplasia which are thought to have a connection with the development of adenocarcinoma and neoplasms, Bahadır et al. ²² detected intestinal metaplasia in 24 of the 351 gallbladders with 11 having epithelial dysplasia of varying degrees. Incidental carcinoma was found in 4 of the 11 cases with intestinal metaplasia and dysplasia. In their study on polypoid lesions, Yıldırım et al. ²³ detected 1 case of adenocarcinoma in 1420 cholecystectomy specimens. In a similar study by Tunçel et al. ^24, 1 adenocarcinoma was reported in 99 polypoid lesions that were found in 4,479 cholecystectomy specimens. Koca et al. ²⁵ reported the incidence of carcinoma, biliary intraepithelial neoplasia and papillary neoplasia as approximately 2%. A similar percentage was also reported by Mazlum et al. ²⁶. Argon et al. ²⁷ recommend sampling a full slice longitudinally all along the gallbladder as this method reveals the presence of a higher number of pyloric metaplasia, intestinal metaplasia, lowgrade dysplasia, and invasive carcinoma than the method of sampling the fundus and the body separately. Bolat et al. ²⁸ stated that the routine method in their pathology laboratory is to take three samples from cholecystectomy specimens. They mapped and examined 75 cholecystectomy specimens and reported that they observed an increase in the incidence of metaplasia, dysplasia, and early carcinoma detection when the number of samplings was increased.

The adenoma-carcinoma sequence has also been extensively debated recently ². Some authors claim that the adenomacarcinoma sequence is a natural process in adenocarcinoma development ²⁹. There are other studies that support this idea, stating that there is a presence of adenomatous residue in the areas where invasive carcinoma develops ³⁰. There are also other publications of case series where early microcarcinoma development in the area of adenoma was reported ³¹ and there are also certain studies that focus on the adenomas with dysplasia-carcinoma in situinvasive carcinoma development including pyloric gland adenomas ³². However, the results obtained from a comprehensive series study do not support this view ². Intestinal metaplasia has been reported to have a significant correlation with epithelial hyperplasia and dysplasia ¹⁷.

The importance and necessity of specifying each and every lesion encountered in the gallbladder is also another object at issue. It seems that the diagnosis may change if new sections are taken from the specimen when any epithelial abnormality is seen during microscopic examination of the cholecystectomy materials ³. Bivins et al. ³³ assert that 4 sections would be sufficient in detecting most cases of dysplasia. Similarly, it is reported that, in cases where dysplasia or reactive atypia is detected after the routine sampling process, a re-sampling of up to 4 sections cut from the re-examined gallbladder is sufficient in detecting all important lesions and that sampling the entire gallbladder is not necessary ³⁴. In cases of in situ carcinoma/highgrade dysplasia where the suspicion of invasion is excluded and the given lesions are entirely followed up, the clinical course has been reported to be quite good ^35,36. However, criteria for both sampling and diagnosis of low-grade lesions and for the differentiation between reactive atypia and low-grade dysplasia have not been clearly specified yet ³⁷. Pathologists generally feel the need for sampling new sections and extending the investigation mostly when highand low-grade BilIN is present. According to Renshaw and Gould ^34, consultant institutions recommend a reexamination of the cholecystectomy specimen and sampling of the entire gallbladder upon identification of these lesions but this, in accordance with the aforementioned study, is not a necessary and appropriate approach in order to reach a definitive diagnosis. Regarding the stance of these authors, in a short article written by Adsay et al. ³ in 2013, it is stated that neoplastic lesions are not easily identified, particularly in macroscopic examination of the gallbladder samples, and the risk in the population should also be taken into account in these terms. It is also emphasized that the relative coexistence of epithelial lesions in the gallbladder with neoplastic lesions should be kept in mind. Regarding this issue, the decision taken in the consensus meeting sponsored by Americas Hepato-Pancreato-Biliary Association in 2015 asserts that in high incidence areas, sampling of the routine cholecystectomy specimens should include a minimum of three sections representing the wall of the bladder and the cystic duct margin; and that the specimens with dysplasia or cancer should be entirely sampled ³⁸.

As proven above, there are various methods and recommendations in the literature and reference books for surgical pathology regarding the procedure for the sampling of cholecystectomy specimens.

In conclusion, although the percentage of invasive carcinoma in all cholecystectomy specimens examined in our study is compatible with the ones in the literature, the rates of metaplasias and neoplasms are different and also the incidence rate of the lesions differ from one institution to another. The major causes of this difference include the differences in the frequency of oncological surgery among the institutions, non-uniformity of the macroscopic sampling protocols, geographical dissimilarities, and the differences in the diagnoses/interpretations of the pathologists. Besides, during the routine pathology practices, pathologists do not feel the need for reporting some epithelial changes/lesions identified in the cholecystectomy specimens (i.e. antral and intestinal metaplasia).

It is necessary for putting an end to this confusion to identify the accurate incidence rate of the gallbladder lesions in Turkey, design a common path for the standardisation of reporting with uniformity in diagnosis/terminology and to conduct more studies including different geographical regions by developing a new standardised method of macroscopic sampling and microscopic examination. In accordance with the results obtained, new sampling criteria were specified and a prospective multicentre study has been commenced with the aim of identifying the accurate incidence rate of gallbladder lesions, standardising the reporting process, and promoting uniformity in the diagnosis/terminology according to these prescribed methods of macroscopic sampling and microscopic examination.

CONFLICT of INTEREST
The authors declare no conflict of interest.

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