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- Materials and Methods
Acinar cell carcinomas (ACCs) of the pancreas are characterized by the histological and immunohistochemical features of acinar cell differentiation. Recently, BCL10, originally identified as a recurrent t(1;14)(p22;q32) translocation in MALT B-cell lymphoma, was found to be immunohistochemically positive in some solid tumors, including ACC. To evaluate its diagnostic efficacy, we performed BCL10 immunohistochemistry and evaluated molecular markers correlated to pancreatic tumor lineages (neuroendocrine markers and a mutation analysis of KRAS and GNAS) using samples from 126 pancreatic tumors (17 ACCs, 24 pancreatic ductal adenocarcinomas, 4 adenosquamous carcinomas, 9 intraductal papillary mucinous neoplasms, 10 mucinous cystic neoplasms, 44 neuroendocrine tumors, 9 serous cystic tumors and 10 solid-pseudopapillary neoplasms). BCL10 was exclusively expressed in normal acini. In pancreatic tumors, 14 of 17 (82%) ACCs and 2 of 4 (50%) adenosquamous carcinomas were positive, while the other subtypes were almost negative. We subsequently examined the diagnostic utility of BCL10 in endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimens using 57 pancreatic tumors. BLC10 correctly identified ACCs (9/13) and adenosquamous carcinomas (2/4) but none of the other subtypes (n = 41). Therefore, we suggested that BCL10 expression is a useful marker for acinar cell differentiation, particularly in the diagnosis of EUS-FNA specimens.
Pancreatic ductal adenocarcinoma (PDA) is a major subtype of pancreatic cancer that is followed by neuroendocrine tumors. These two major subtypes represent the cellular composition of the pancreas. Although most other neoplasms are associated with ductal epithelial differentiation, i.e. intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), some tumors show acinar features. Pancreatic acinar cell carcinoma (ACC) is a tumor characterized by acinar cell features and comprises less than 2% of all pancreatic neoplasms.[1, 2] This subtype is clinically and pathologically distinct from typical PDAs. The median age of the patients with ACC is slightly older than PDA, and ACC shows a better prognosis despite more frequent distant metastasis. Histologically, ACC has characteristic acinar differentiation that is illustrated by immunohistochemical staining for trypsin, chymotrypsin, and/or lipase.[3-5] Furthermore, ACCs have been reported to lack a KRAS mutation, suggesting that they develop via different molecular pathways than PDAs. Abnormalities in tumor suppressor genes, such as TP53, DPC4/Smad4 and p16, are less common than in PDAs.[3, 6-9]
BCL10 was recently identified through the cloning of a (1;14)(p22;q32) translocation breakpoint in several cases of low-grade mucosa-associated lymphoid tissue (MALT) B-cell lymphoma.[10, 11] This gene, localized to chromosome band 1p22, is a cellular homolog of the equine herpesvirus-2 E10 gene; both contain an amino-terminal caspase recruitment domain (CARD) homologous to that found in several apoptotic molecules. Mutation analyses of BCL10 have implicated this gene in MALT lymphomas and other lymphoid tumors of the B- or T-cell lineage without t(1;14)(p22;q32) translocation.[13-15] In addition, BCL10 abnormalities have been reported in solid cancer cell lines and tumors, including malignant mesotheliomas, germ cell tumors, and colon carcinomas, suggesting that BCL10 can contribute to the pathogenesis of several types of neoplasia.[10, 16] In contrast to a number of articles about BCL10 expression in lymphoid malignancies, only a limited number of articles have documented its expression in solid cancers. Chang et al. have reported that BCL10 expression is significantly associated with the progression and prognosis of oral squamous cell carcinomas, while BCL10 has been reported by Kuo et al. to play an important role in controlling the growth of cervical cancer cells through NF-κB-dependent cyclin D1 regulation.[17, 18] In the pancreas, La Rosa et al. recently reported that BCL10 was expressed specifically in acinar cells and acinar cell carcinomas.[5, 19]
Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) was introduced into clinical practice in the early 1990s and is now considered one of the most useful methods for the histological diagnosis and staging of pancreatic cancers. However, specimens obtained by EUS-FNA are tiny and fragmented, and a definitive diagnosis is frequently challenging for pathologists. We recently reported a diagnostic scheme for EUS-FNA specimens of three major pancreatic tumor types using a minimal number of markers, including CK7, CDX2, synaptophysin, chromogranin A and KRAS mutations. In that study, ACC was characterized by occasional expression of CK7 and CDX2, lack of a KRAS mutation, and various expression patterns of neuroendocrine markers. When heterogeneous expression and staining errors were considered, it was determined that some positive markers were required in this panel. In the present study, we found that BCL10 is expressed exclusively in ACC, implying that it could serve as a useful marker for labeling this rare, well-differentiated subtype, particularly when diagnosing EUS-FNA specimens.
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- Materials and Methods
Various cellular components are present in the pancreas, and the current classification of pancreatic cancer is based on the knowledge of such normal counterparts. ACC is thought to be derived from pancreatic acinar cells, and ACC mimics the morphology and phenotype of the normal counterpart, acinar cells, including trypsin expression. However, ACC is frequently positive for neuroendocrine makers, despite early divergence in the development of the pancreas. As shown in Tables 1 and 2, approximately one third of ACCs expressed neuroendocrine markers. Furthermore, both tumors shared morphological characteristics including small round nuclei, central localization of the nuclei in the cytoplasm, relative ample cytoplasm, and sheet-like growth. These morphological characteristics, in addition to the frequent expression of neuroendocrine markers, make the differential diagnosis between ACC and neuroendocrine tumor difficult. However, this study clearly demonstrated that BCL10 could be used in this setting. None of the 44 neuroendocrine tumors, which included three cases of poorly differentiated neuroendocrine carcinomas, were positive for BCL10, but 82% (14/17) of ACCs expressed BCL10. This specific expression is especially useful for EUS-FNA samples because only a tiny piece of the tissues is allowed to be examined, and in many cases, no normal acini are included for control staining. Indeed, the commonly used ACC markers, such as trypsin and chymotrypsin, sometimes stained weakly and focally. Even though overall frequency of positive trypsin was higher than that of BCL10, distinctively positive reactions (2+ and 3+ in Table 2) were limited to only one third of ACCs. Furthermore, faint reaction (1+) in two surgical specimens was not detected in the EUS-FNA samples. This is in sharp contrast to BCL10, in which we found clear labeling (all 2+ or 3+) in EUS-FNA samples of ACCs. Such clear reaction is particularly crucial for diagnosis with EUS-FNA samples. Although two articles from the same group have reported specific expression of BCL10 in ACC using surgical specimens,[5, 19] we confirmed the finding with detailed genotypes, and found that the expression was useful particularly in diagnosis using EUS-FNA samples.
In addition to neuroendocrine tumors, ACC with prominent ductal differentiation may be problematic in differential diagnoses. Stelow et al. have recently reported 11 such cases, five and six of which showed some morphological features of mucinous carcinoma and typical ductal carcinoma, respectively. For this type of tumor, a differential diagnosis with PDA, IPMN and MCN is needed. Fortunately, good genetic markers are available: KRAS and GNAS. KRAS mutations were previously detected in 95% or more of PDAs, 40–80% of IPMNs, and 30% of MCNs, while GNAS mutations were specific to IPMNs.[23-27] This study revealed that BCL10 was expressed in a mutually exclusive fashion to KRAS and GNAS mutations, suggesting that BCL10-expressing tumors are distinct from PDAs, IPMNs and MCNs.
It is also interesting that adenosquamous carcinomas expressed BCL10, although the number of examined samples was limited. Prior to this study, we examined BCL10 expression in 130 tumors of various organs using tissue microarray (data not shown, but could be provided as supplementary data if requested). Five of 33 squamous carcinomas but no adenocarcinomas were positive for BCL10. Indeed, some reports have noted that BCL10 is expressed in most oral squamous cell carcinomas[17, 30] and that the intensity is associated with cancer progression and prognosis. Therefore, squamous cell differentiation could be a pitfall of the interpretation of BCL10 expression in pancreatic tumor subtyping.
In summary, we examined BCL10 expression in pancreatic cancer. BCL10 was specifically expressed in ACCs, and none of the BCL10-expressing tumors harbored the KRAS or GNAS mutations that are frequently mutated in PDAs, IPMNs and MCNs. ACC is often difficult to distinguish from neuroendocrine tumors, particularly when limited samples are obtained, because approximately one third of ACCs are positive for neuroendocrine markers and the two tumors share morphological characteristics. BCL10 could be a useful marker in this setting.