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Perinodular CK19 loss in hepatocarcinogenesis
Clinics and Research in Hepatology and Gastroenterology (2011) 35, 783—785
COMMENTARY
Perinodular CK19 loss in hepatocarcinogenesis Paulette Bioulac-Sage , Charles Balabaud ∗ GREF Inserm U1053, service d’anatomie pathologique, hôpital Pellegrin, CHU de Bordeaux, université Bordeaux-Segalen, 33076 Bordeaux cedex, France Available online 1st October 2011
Summary Cirrhotic nodules containing hepatocytes are surrounded by perinodular stroma, that consists of an expanded fibrous matrix and epithelial cells with ductular phenotype, the ‘‘ductular-reaction’’. Stromal invasion is a key histopathologic feature used to differentiate premalignant dysplastic nodules from malignant hepatocellular carcinoma (HCC). K19 immunoreactivity in the stromal compartment in cirrhotic explants was examined. Quantitative differences were manifested in three distinct histologically identifiable patterns: ‘‘complex’’ around cirrhotic nodules, ‘‘attenuated’’ around dysplastic nodules, and ‘‘absent’’ around HCC. These findings suggest marked alterations in cellular identity as an underlying mechanism for the reproducible extralesional K19 pattern that parallels progressive stages of intranodular hepatocarcinogenesis. Paracrine signalling is proposed as a link that emphasizes the importance of the epithelial-stromal compartment in malignant progression of HCC in cirrhosis. © 2011 Published by Elsevier Masson SAS.
Cirrhotic nodules (CN) containing hepatocytes are surrounded by perinodular stroma, that consists of an expanded fibrous matrix harboring lymphovascular structures, mixed inflammatory cells, fibrogenic cells and epithelial cells with ductular phenotype, the ‘‘ductular-reaction’’ (DR). The presence of these biliary-type epithelial cells between mesenchymal elements makes perinodular cirrhotic septa an epithelial-stromal compartment (Fig. 1). Stromal invasion is a key histopathologic feature used to differentiate premalignant dysplastic nodules from malignant hepatocellular carcinoma [1]. Lack of the DR in the stromal compartment around hepatocellular carcinoma (HCC) has been documented [2]. However, the relationship of epithelial keratin 19 (K19) structures to progression of
∗
Corresponding author. E-mail addresses: [email protected] (P. Bioulac-Sage), [email protected] (C. Balabaud).
intralesional carcinogenesis has not been explored. The aim of the present study performed by the team of Brunt (St Louis USA) was to correlate perinodular K19 patterns with the intranodular hepatocellular pathologic alterations, and to quantify a relationship of intranodular and extranodular findings [3]. On selected nodules from liver explants established diagnostic criteria were applied to identify cirrhotic nodules (CN), dysplastic nodules (DN), and hepatocellular carcinoma (HCC). Necrosis was defined using established criteria. Apoptosis was assessed using morphologic features, immunostaining for activated caspase 3, and detection of DNA fragmentation and chromatin cleavage using TUNEL. Cellular phenotype (epithelial [Ep] versus mesenchymal [Me]) was defined via a combination of topographic, architectural, cytomorphologic, nuclear, and immunophenotypic features adapted from proposed criteria. These markers included transforming growth factor-1-receptor (TGF1R), fibroblast-specific protein 1 (FSP-1), Vimentin,
2210-7401/$ – see front matter © 2011 Published by Elsevier Masson SAS. doi:10.1016/j.clinre.2011.08.006
784
P. Bioulac-Sage, C. Balabaud
Figure 1
Ductular reaction around cirrhotic and dysplastic nodules.
SNAIL, different SMADs, and E-cadherin. Cellular identity alteration was defined as discrepancies in these features at the cellular level. Authors focused on (TGF-) because of its major profibrogenic role, established implications in human biliary epithelial mesenchymal transition (EMT), and signaling upstream of SNAIL via recruitment of SMAD molecules. The cohort consisted of 170 nodules: 71 CN, 45 DN, and 54 HCC. Specifically, the DR around CN demonstrated numerous ductules consisting of interlacing epithelial clusters that blended into the hepatocyte interface. Three arbitrary zones could be distinguished (Fig. 1): • central regions of the internodular septa contained larger ductules, often interspersed with thin-walled vessels; • the perinodular zone, in which a mixture of ring-shaped ductules and small circular epithelial groups, with or without a central lumen, predominates and; • an interface in which slender strings of biliary-type epithelial cells are located between hepatocytes and merge into HCN, resulting in a radial or spoke-like appearance with orientation of cells perpendicular to the perimeter of the nodule. These features appreciated using K19 labeling was termed ‘‘complex’’. Depletion of ductules with lumina was termed attenuated (Fig. 1). The stroma around welldifferentiated HCC was depleted of biliary-type epithelial elements, and the DR and K19 immunoreactivity was ‘‘absent’’. Three patterns of DR surrounding CN, DN, and HCC, i.e., complex, attenuated, and absent, respectively, were reproducibly documented. The highest prevalence of the complex pattern was observed around CN, the attenuated pattern around DN, and the absent pattern around HCC. The complex perinodular K19 pattern was not observed around HCC. Interestingly
enough the perinodular K19 pattern can function as an accurate surrogate for intranodular disease. Density of cells and profiles was expressed per sector or micrometer squared, and statistical comparison demonstrated a highly significant reduction in K19-positive elements from CN to DN to HCC. Fewer cells contribute to perinodular profile counts in the progressive stages of hepatocarcinogenesis and support the morphologic rather than temporal descriptors complex, attenuated, and absent. Assessment of features of both necrosis and apoptosis argue against cell death as the predominant morphologic mechanisms contributing to the documented perinodular K19 loss. Multicolor immunolabeling for K19, vimentin, ECADH, SNAIL, and fibroblast-specific protein 1 (FSP-1) demonstrated discrepancies in immunophenotype and cytomorphologic features. Variability of cellular features was accompanied by an overall decrease in epithelial markers and significantly increased fractions of SNAIL and FSP-1positive cells in the DR around DN when compared with CN (P < 0.0001). Immunolabeling of transforming growth factor signaling components (TGF1R, SMAD3, and pSMAD2/3) demonstrated increased percentages of pSMAD2/3 around DN when compared with CN (P < 0.0001). These findings collectively suggest marked alterations in cellular identity as an underlying mechanism for the reproducible extralesional K19 pattern that parallels progressive stages of intranodular hepatocarcinogenesis. The authors proposed paracrine signaling across the lesional interface as a possible underlying pathophysiologic link that emphasizes the importance of the epithelialstromal compartment in malignant progression of HCC in cirrhosis. This study describes and further interrogates for the first time a compelling relationship of the extralesional K19 compartment that parallels intralesional malignant
K19 loss transformation in progressive stages of hepatocarcinogenesis in cirrhosis. In particular, K19 loss follows distinct morphologic patterns, with a complex pattern around CN, an attenuated configuration around DN (Fig. 1), and absence around HCC. These findings emphasize alterations of an intricate relationship between intralesional and extralesional compartments as hepatocarcinogenesis progresses. Changes in the nonhepatocellular epithelial compartment remain largely unexplored, and these findings provide a morphologic starting point to examine whether loss of perinodular DR is a bystanding or contributing factor in progressive stages of hepatocarcinogenesis. Additional data will be necessary to implement these interesting and novel findings. Briefly, definitive proof that perinodular K19-positive cells are able to transdifferentiate into mesenchymal elements would require in vivo lineage tracing with cholangiocyte-specific marker genes; the hypothesis of paracrine signaling does not exclude additional contributing mechanisms such as non-TGF--mediated triggers. It remains also to be determined whether perinodular K19 assessment can overcome the recognized limitations associated with needle core biopsy sampling. Finally it might be of interest to analyse separately low and high
785 grade dysplasia nodules particularly if, as demonstrated, perinodular K19 patterns can function as a highly reliable extralesional surrogate for intralesional disease, with sensitivity and specificity values well above 83%. Perinodular K19 patterns will not be observed in HCC developed on noncirrhotic liver and in early stages of cirrhosis.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
References [1] Kojiro M. Pathological diagnosis at early stage: reaching international consensus. Oncology 2010;78Suppl1:31—5. [2] Park YN, Kojiro M, Di Tommaso L, Dhillon AP, Kondo F, Nakano M, et al. Ductular reaction is helpful in defining early stromal invasion, small hepatocellular carcinomas, and dysplastic nodules. Cancer 2007;109:915—23. [3] Lennerz JKM, Chapman WC, Brunt EM. Keratin 19 epithelial patterns in cirrhotic stroma parallel hepatocarcinogenesis. Am J Pathol 2011;179:1015—29.
COMMENTARY
Perinodular CK19 loss in hepatocarcinogenesis Paulette Bioulac-Sage , Charles Balabaud ∗ GREF Inserm U1053, service d’anatomie pathologique, hôpital Pellegrin, CHU de Bordeaux, université Bordeaux-Segalen, 33076 Bordeaux cedex, France Available online 1st October 2011
Summary Cirrhotic nodules containing hepatocytes are surrounded by perinodular stroma, that consists of an expanded fibrous matrix and epithelial cells with ductular phenotype, the ‘‘ductular-reaction’’. Stromal invasion is a key histopathologic feature used to differentiate premalignant dysplastic nodules from malignant hepatocellular carcinoma (HCC). K19 immunoreactivity in the stromal compartment in cirrhotic explants was examined. Quantitative differences were manifested in three distinct histologically identifiable patterns: ‘‘complex’’ around cirrhotic nodules, ‘‘attenuated’’ around dysplastic nodules, and ‘‘absent’’ around HCC. These findings suggest marked alterations in cellular identity as an underlying mechanism for the reproducible extralesional K19 pattern that parallels progressive stages of intranodular hepatocarcinogenesis. Paracrine signalling is proposed as a link that emphasizes the importance of the epithelial-stromal compartment in malignant progression of HCC in cirrhosis. © 2011 Published by Elsevier Masson SAS.
Cirrhotic nodules (CN) containing hepatocytes are surrounded by perinodular stroma, that consists of an expanded fibrous matrix harboring lymphovascular structures, mixed inflammatory cells, fibrogenic cells and epithelial cells with ductular phenotype, the ‘‘ductular-reaction’’ (DR). The presence of these biliary-type epithelial cells between mesenchymal elements makes perinodular cirrhotic septa an epithelial-stromal compartment (Fig. 1). Stromal invasion is a key histopathologic feature used to differentiate premalignant dysplastic nodules from malignant hepatocellular carcinoma [1]. Lack of the DR in the stromal compartment around hepatocellular carcinoma (HCC) has been documented [2]. However, the relationship of epithelial keratin 19 (K19) structures to progression of
∗
Corresponding author. E-mail addresses: [email protected] (P. Bioulac-Sage), [email protected] (C. Balabaud).
intralesional carcinogenesis has not been explored. The aim of the present study performed by the team of Brunt (St Louis USA) was to correlate perinodular K19 patterns with the intranodular hepatocellular pathologic alterations, and to quantify a relationship of intranodular and extranodular findings [3]. On selected nodules from liver explants established diagnostic criteria were applied to identify cirrhotic nodules (CN), dysplastic nodules (DN), and hepatocellular carcinoma (HCC). Necrosis was defined using established criteria. Apoptosis was assessed using morphologic features, immunostaining for activated caspase 3, and detection of DNA fragmentation and chromatin cleavage using TUNEL. Cellular phenotype (epithelial [Ep] versus mesenchymal [Me]) was defined via a combination of topographic, architectural, cytomorphologic, nuclear, and immunophenotypic features adapted from proposed criteria. These markers included transforming growth factor-1-receptor (TGF1R), fibroblast-specific protein 1 (FSP-1), Vimentin,
2210-7401/$ – see front matter © 2011 Published by Elsevier Masson SAS. doi:10.1016/j.clinre.2011.08.006
784
P. Bioulac-Sage, C. Balabaud
Figure 1
Ductular reaction around cirrhotic and dysplastic nodules.
SNAIL, different SMADs, and E-cadherin. Cellular identity alteration was defined as discrepancies in these features at the cellular level. Authors focused on (TGF-) because of its major profibrogenic role, established implications in human biliary epithelial mesenchymal transition (EMT), and signaling upstream of SNAIL via recruitment of SMAD molecules. The cohort consisted of 170 nodules: 71 CN, 45 DN, and 54 HCC. Specifically, the DR around CN demonstrated numerous ductules consisting of interlacing epithelial clusters that blended into the hepatocyte interface. Three arbitrary zones could be distinguished (Fig. 1): • central regions of the internodular septa contained larger ductules, often interspersed with thin-walled vessels; • the perinodular zone, in which a mixture of ring-shaped ductules and small circular epithelial groups, with or without a central lumen, predominates and; • an interface in which slender strings of biliary-type epithelial cells are located between hepatocytes and merge into HCN, resulting in a radial or spoke-like appearance with orientation of cells perpendicular to the perimeter of the nodule. These features appreciated using K19 labeling was termed ‘‘complex’’. Depletion of ductules with lumina was termed attenuated (Fig. 1). The stroma around welldifferentiated HCC was depleted of biliary-type epithelial elements, and the DR and K19 immunoreactivity was ‘‘absent’’. Three patterns of DR surrounding CN, DN, and HCC, i.e., complex, attenuated, and absent, respectively, were reproducibly documented. The highest prevalence of the complex pattern was observed around CN, the attenuated pattern around DN, and the absent pattern around HCC. The complex perinodular K19 pattern was not observed around HCC. Interestingly
enough the perinodular K19 pattern can function as an accurate surrogate for intranodular disease. Density of cells and profiles was expressed per sector or micrometer squared, and statistical comparison demonstrated a highly significant reduction in K19-positive elements from CN to DN to HCC. Fewer cells contribute to perinodular profile counts in the progressive stages of hepatocarcinogenesis and support the morphologic rather than temporal descriptors complex, attenuated, and absent. Assessment of features of both necrosis and apoptosis argue against cell death as the predominant morphologic mechanisms contributing to the documented perinodular K19 loss. Multicolor immunolabeling for K19, vimentin, ECADH, SNAIL, and fibroblast-specific protein 1 (FSP-1) demonstrated discrepancies in immunophenotype and cytomorphologic features. Variability of cellular features was accompanied by an overall decrease in epithelial markers and significantly increased fractions of SNAIL and FSP-1positive cells in the DR around DN when compared with CN (P < 0.0001). Immunolabeling of transforming growth factor signaling components (TGF1R, SMAD3, and pSMAD2/3) demonstrated increased percentages of pSMAD2/3 around DN when compared with CN (P < 0.0001). These findings collectively suggest marked alterations in cellular identity as an underlying mechanism for the reproducible extralesional K19 pattern that parallels progressive stages of intranodular hepatocarcinogenesis. The authors proposed paracrine signaling across the lesional interface as a possible underlying pathophysiologic link that emphasizes the importance of the epithelialstromal compartment in malignant progression of HCC in cirrhosis. This study describes and further interrogates for the first time a compelling relationship of the extralesional K19 compartment that parallels intralesional malignant
K19 loss transformation in progressive stages of hepatocarcinogenesis in cirrhosis. In particular, K19 loss follows distinct morphologic patterns, with a complex pattern around CN, an attenuated configuration around DN (Fig. 1), and absence around HCC. These findings emphasize alterations of an intricate relationship between intralesional and extralesional compartments as hepatocarcinogenesis progresses. Changes in the nonhepatocellular epithelial compartment remain largely unexplored, and these findings provide a morphologic starting point to examine whether loss of perinodular DR is a bystanding or contributing factor in progressive stages of hepatocarcinogenesis. Additional data will be necessary to implement these interesting and novel findings. Briefly, definitive proof that perinodular K19-positive cells are able to transdifferentiate into mesenchymal elements would require in vivo lineage tracing with cholangiocyte-specific marker genes; the hypothesis of paracrine signaling does not exclude additional contributing mechanisms such as non-TGF--mediated triggers. It remains also to be determined whether perinodular K19 assessment can overcome the recognized limitations associated with needle core biopsy sampling. Finally it might be of interest to analyse separately low and high
785 grade dysplasia nodules particularly if, as demonstrated, perinodular K19 patterns can function as a highly reliable extralesional surrogate for intralesional disease, with sensitivity and specificity values well above 83%. Perinodular K19 patterns will not be observed in HCC developed on noncirrhotic liver and in early stages of cirrhosis.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
References [1] Kojiro M. Pathological diagnosis at early stage: reaching international consensus. Oncology 2010;78Suppl1:31—5. [2] Park YN, Kojiro M, Di Tommaso L, Dhillon AP, Kondo F, Nakano M, et al. Ductular reaction is helpful in defining early stromal invasion, small hepatocellular carcinomas, and dysplastic nodules. Cancer 2007;109:915—23. [3] Lennerz JKM, Chapman WC, Brunt EM. Keratin 19 epithelial patterns in cirrhotic stroma parallel hepatocarcinogenesis. Am J Pathol 2011;179:1015—29.