Mutational profiling and immunohistochemical analysis of a surgical series of ampullary carcinomas.

AIMS: Knowledge regarding the genetic features of ampullary carcinoma (AC) in European patients is limited. The utility of tumour markers for the establishment of a malignant diagnosis in biopsies from the ampullary region has not been fully elucidated. We aimed to describe the clinical, pathological, immunohistochemical (IHC) and genetic features of a Danish series of surgically resected ACs. METHODS: Surgically resected ACs (n=59) were examined regarding (1) clinicopathological features, (2) histological subtypes, (3) expression of IMP3, maspin, MUC5AC and S100P and (4) next-generation sequencing using a hybrid capture-based platform (Illumina HiSeq2500), including 315 cancer-related genes plus introns from 28 genes often rearranged or altered in cancer. Tumour mutational burden (TMB) and microsatellite instability (MSI) were also evaluated. RESULTS: Pancreatobiliary adenocarcinomas (PB-AC), intestinal adenocarcinomas (INT-AC), other ampullary tumours and mixed adenocarcinomas represented 45.8%, 23.7%, 16.9% and 13.6%. The proportion of IHC-positive ACs (score ≥2) was: Maspin (94.9%), IMP3 (67.8%), S100P (39.0%) and MUC5AC (18.6%). Most frequently altered genes were TP53 (59.3%), KRAS (40.7%), APC (27.8%), SMAD4 (20.4%), CDKN2A (16.7%) and ARID2/PIK3CA (each 11.1%). MUC5AC and S100P were frequently expressed in PB-AC, APC alterations frequent in INT-AC, SOX9 alterations were exclusive in INT-AC and MDM2 and FRS2 alterations in PB-AC. Four of 49 ACs (8.2%) were TMB-high/MSI-high and showed loss of MLH1 and PMS2. CONCLUSIONS: PB-AC was the most frequent histological subtype of AC. Maspin and IMP3 were the IHC tumour markers with the highest sensitivity. Adenocarcinoma subtypes differed regarding several genetic alterations, whose predictive value remains to be evaluated.

Mixed Adenoma Well-differentiated Neuroendocrine Tumor (MANET) of the Digestive System: An Indolent Subtype of Mixed Neuroendocrine-NonNeuroendocrine Neoplasm (MiNEN).

Mixed neuroendocrine-nonneuroendocrine neoplasm (MiNEN) consisting of adenoma and well-differentiated neuroendocrine tumor (NET) has been recently defined as "MANET." However, the clinico-pathologic and pathogenetic features of this entity are not thoroughly studied. We examined the clinico-pathologic features of 12 MANETs by expanding their p53 and ß-catenin expression profiles as well as the presence of microsatellite instability and KRAS, BRAF, and PIK3CA mutations in the 2 tumor components. In all cases, the adenomatous component represented the larger part of the lesions and the NET was localized in the deep central portion of polyps. In 9 cases the latter was represented by NET G1, in 2 by NET G2, and in 1 by NET G3. In all cases, the glandular and NET components were intimately admixed, with zone of transition between the tumor components. The NET component was p53 negative in all cases and 3 of 8 cases showed variable nuclear positivity for ß-catenin. All patients with follow-up data were alive and free of disease after a mean follow-up time of 9 years. No mutations in KRAS, BRAF, and PIK3CA genes and no microsatellite instability were found in both tumor components. Review of the literature also identified 59 previously reported MANETs and no tumor-related death has been found. Like mixed adenoneuroendocrine carcinomas, a high-grade malignant form of MiNENs with a poorly differentiated neuroendocrine carcinoma component, a common origin for both tumor constituents may be hypothesized. Moreover, the current series provides evidence that MANET is an indolent disease that needs to be distinguished from aggressive high-grade MiNENs.

Cutaneous Metastasis of Adenocarcinoma of the Ampulla of Vater.

Cutaneous metastases from an adenocarcinoma of the Ampulla of Vater are very rare, with only a few cases previously reported. We present here an additional case in a 57-year-old woman who complained of a painful growth on her frontal scalp that she had noticed 4 months earlier. Her medical history included an ampullary adenocarcinoma, which was diagnosed 4 years ago, excised through a Whipple procedure, and treated using chemotherapy and radiotherapy. The scalp biopsy showed a dermal and epidermotropic well-differentiated glandular neoplasm with abundant neutrophils within the luminae of the tumoral glands. The tumor failed to express p63 and cytokeratin 5/6, whereas it was intensively positive for CK7 and E-cadherin. CDX2 expression was weak and focal. The immunohistochemical expression of DNA mismatch-repair proteins (MSH2, MSH6, MLH1, and PMS2) was preserved. Despite oncological treatment, the patient developed multiple cutaneous metastases during the ensuing several months, and eventually died 6 years after her initial diagnosis with widespread metastases.

Neural and neurogenic tumours of the gastroenteropancreaticobiliary tract.

Neural lesions occur uncommonly in the gastroenteropancreaticobiliary tract. However, due to the growing number of screening colonoscopy procedures, polypoid neural lesions of the colon are being recognised increasingly and range from benign tumours to high-grade malignant neoplasms. Morphological variability of neural tumours can be wide, although some entities share pathological features, and, as such, these lesions can be diagnostically challenging. We review the spectrum of pathology of neural tumours in the gastroenteropancreaticobiliary tract, with the goal of providing a practical approach for practising surgical pathologists.

Treponema denticola chymotrypsin-like proteinase may contribute to orodigestive carcinogenesis through immunomodulation.

BACKGROUND: Periodontal pathogens have been linked to oral and gastrointestinal (orodigestive) carcinogenesis. However, the exact mechanisms remain unknown. Treponema denticola (Td) is associated with severe periodontitis, a chronic inflammatory disease leading to tooth loss. The anaerobic spirochete Td is an invasive bacteria due to its major virulence factor chymotrypsin-like proteinase. Here we aimed to investigate the presence of Td chymotrypsin-like proteinase (Td-CTLP) in major orodigestive tumours and to elucidate potential mechanisms for Td to contribute to carcinogenesis. METHODS: The presence of Td-CTLP within orodigestive tumour tissues was examined using immunohistochemistry. Oral, tonsillar, and oesophageal squamous cell carcinomas, alongside gastric, pancreatic, and colon adenocarcinomas were stained with a Td-CTLP-specific antibody. Gingival tissue from periodontitis patients served as positive controls. SDS-PAGE and immunoblot were used to analyse the immumodulatory activity of Td-CTLP in vitro. RESULTS: Td-CTLP was present in majority of orodigestive tumour samples. Td-CTLP was found to convert pro MMP-8 and -9 into their active forms. In addition, Td-CTLP was able to degrade the proteinase inhibitors TIMP-1, TIMP-2, and α-1-antichymotrypsin, as well as complement C1q. CONCLUSIONS: Because of its presence within tumours and regulatory activity on proteins critical for the regulation of tumour microenvironment and inflammation, the Td-CTLP may contribute to orodigestive carcinogenesis.

Expression of PD-1 and PD-L1 in poorly differentiated neuroendocrine carcinomas of the digestive system: a potential target for anti-PD-1/PD-L1 therapy.

Poorly differentiated neuroendocrine carcinoma of the digestive system has a dismal prognosis with limited treatment options. This study aimed to investigate expression of the PD-1/PD-L1 pathway in these tumors. Thirty-seven patients with a poorly differentiated neuroendocrine carcinoma of the digestive system were identified. Their electronic medical records, pathology reports, and pathology slides were reviewed for demographics, clinical history, and pathologic features. Tumor sections were immunohistochemically labeled for PD-1 and PD-L1, and expression of PD-1 and PD-L1 on tumor and tumor-associated immune cells was analyzed and compared between small cell and large cell neuroendocrine carcinomas. The mean age of patients was 61 years old with 18 men and 19 women. The colorectum (n=20) was the most common primary site; other primary sites included the pancreaticobiliary system, esophagus, stomach, duodenum, and ampulla. Expression of PD-1 was detected on tumor cells (n=6, 16%) as well as on tumor-associated immune cells (n=23, 63%). The 6 cases with PD-1 expression on tumor cells also had the expression on immune cells. Expression of PD-L1 was visualized on tumor cells in 5 cases (14%) and on tumor-associated immune cells in 10 cases (27%). There was no difference in PD-1 and PD-L1 expression between small cell and large cell neuroendocrine carcinomas. In conclusion, PD-1/PD-L1 expression is a frequent occurrence in poorly differentiated neuroendocrine carcinomas of the digestive system. Checkpoint blockade targeting the PD-1/PD-L1 pathway may have a potential role in treating patients with this disease.

Silencing of UCHL1 by CpG promoter hyper-methylation is associated with metastatic gastroenteropancreatic well-differentiated neuroendocrine (carcinoid) tumors.

BACKGROUND: Well-differentiated gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare tumors with varying metastatic potential. The underlying molecular basis for metastasis by GEP-NETs remains undefined. METHODS: Quantitative PCR and immunohistochemistry (IHC) staining for ubiquitin carboxyl-terminal esterase L1 (UCHL1) gene and protein expression was performed on a group of localized and metastatic well-differentiated GEP-NET samples acquired from a prospectively maintained tissue bank. The ability of extent of UCHL1 IHC staining to differentiate localized and metastatic tumors was compared with Ki-67 index. RESULTS: Among 46 total samples, UCHL1 expression at both the gene and protein level was significantly greater among localized GEP-NETs compared with metastatic tumors and metastases (p < 0.001). Hypermethylation of the UCHL1 promoter was commonly observed among metastatic primary tumors and metastases (those with the lowest UCHL1 expression) but not among localized tumors (p < 0.001). Poor staining (<50 %) for UCHL1 was observed in 27 % of localized tumors compared with 87 % of metastatic tumors (p = 0.001). The presence of <50 % staining for UCHL1 was 88 % sensitive and 73 % specific for identifying metastatic disease. In contrast, there was no association between Ki-67 index and metastatic disease. In multivariable analysis, only UCHL1 staining <50 % [odds ratio (OR) 24.5, p = 0.035] and vascular invasion (OR 38.4, p = 0.03) were independent risk factors for metastatic disease at the time of initial surgery. CONCLUSIONS: Loss of UCHL1 expression by CpG promoter hypermethylation is associated with metastatic GEP-NETs. Extent of UCHL1 staining should be explored as a potentially clinically useful adjunct to Ki-67 index in evaluating GEP-NETs for aggressive features.

Cadherin 17 is a novel diagnostic marker for adenocarcinomas of the digestive system.

Cadherin 17 is a member of a multigene family of calcium-dependent, transmembrane proteins that mediates cell-cell adhesion, plays important roles during embryogenesis, and is crucial for tissue morphogenesis and maintenance. Cadherin 17 is exclusively expressed in the epithelial cells of embryonic and adult small intestine and colon, and pancreatic ducts. It has also been reported to be frequently expressed in adenocarcinomas arising in the gastrointestinal tract and pancreas. Owing to its restricted expression in these groups of tumors, cadherin 17 has proven to be a useful immunohistochemical marker for assisting in distinguishing these neoplasms from other malignancies with which they may be confused.

EZH2, a unique marker of malignancy in effusion cytology.

Distinguishing reactive mesothelial cells from metastatic disease, typically adenocarcinoma, in effusion cytology can be challenging at times. We currently use a panel of immunocytochemical markers for select cases including MOC-31 and BerEp4, but difficulties still exist. Enhancer of zeste homologue 2 (EZH2) plays important roles in epigenetic silencing and cell cycle regulation and is upregulated in a wide variety of malignancies. Thus, we hypothesized that EZH2 immunocytochemistry, which to our knowledge has not yet been reported on cytology material, might serve as a unique marker of malignancy in morphologically equivocal effusion specimens by highlighting aberrant protein expression in malignant cells. A total of 96 (48 benign and 48 malignant) effusion cases were selected retrospectively from our department archives. All malignant cases were metastatic adenocarcinomas except for three high grade neuroendocrine carcinomas (two lungs and one ovary), one cervical squamous cell carcinoma, and one epithelioid endometrial stromal sarcoma. The 48 benign cases were all negative for EZH2, and 43 of 48 malignant effusions were positive. As a solitary marker, EZH2 exhibited a sensitivity of 90% and a specificity of 100% (P < 0.0001). EZH2 functioned as a unique and accurate marker of malignancy in this series of effusions. Relative to published data, EZH2 demonstrated a sensitivity comparable to MOC-31 and superior to BerEp4, and a specificity superior to both of these commonly used immunostains. Thus, EZH2 is likely to be of great value as an adjunct to morphology in diagnosing malignancy in effusion specimens.

An invasive extragastrointestinal stromal tumor curably resected following imatinib treatment.

Extragastrointestinal stromal tumors (EGISTs) are rare tumors located outside the gastrointestinal tract. While curable resection is accepted as a noninvasive EGIST treatment, the therapeutic strategy for invasive EGISTs has not yet been established. The present report is the first to show a case of invasive EGIST completely resected after downsizing the tumor with imatinib treatment. A 69-year-old female had multiple masses adjacent to the stomach and ileocecum. The primary lesion measured 18 cm in size and had invaded the stomach, pancreas and liver. The histological findings of fine-needle aspiration samples revealed a proliferation of dysplastic spindle cells that exhibited immunoreactivity for anti-c-kit antibodies. The masses were therefore diagnosed as multiple GISTs with invasion to other organs, with origin difficult to determine at the time. Nineteen months after the imatinib treatment, the tumors were downsized and distinct from the stomach, pancreas and liver. Accordingly, the tumors were regarded to be EGISTs derived from the mesentery. Because they slightly regressed 26 months after treatment, surgery was applied to remove the EGISTs. The intraoperative findings showed no invasive signs, and the tumors were completely removed. The histological findings revealed the presence of dysplastic and c-kit-positive spindle cells in the tumor with an MIB-I index of more than 5%, resulting in a final diagnosis of high-risk EGIST derived from the mesentery. No recurrence was detected for 16 months after resection. In conclusion, preoperative treatment with imatinib followed by curable resection is a feasible option to cure invasive EGISTs.

The pitfalls of CA19-9: routine testing and comparison of two automated immunoassays in a reference oncology center.

We evaluated CA19-9 as a marker of various malignancies and compared the results of 2 commercial immunoassays. The Abbott ARCHITECT i2000 and Roche cobas 410 immunoassays were used on 500 consecutive samples to evaluate the frequency of positive results by cancer type and the correlation between assays. The patients were tested before or after surgery and/or during chemotherapy. The rate of results exceeding conventional thresholds was 92.3% in pancreatic cancer, 36.8% in gastric cancer, and ranged from 3.0% to 35.9% in other tumors. Agreement (90.6%) and correlation (R(2) = 0.865) between the 2 assays were good and the frequency of highly discordant results was low (6/500). In some cases, interference by heterophilic antibodies was demonstrated. The 2 methods were comparable in diagnostic accuracy and had good correlation but are not interchangeable. Patients should always be monitored for CA19-9 with the same method and it should be indicated in the report.

Prognoses of GEP-NETS with undetermined malignant potentials of their primary sites.

BACKGROUND/AIMS: In gastroenteropancreatic neuroendocrine tumors (GEP-NETs), primary lesions cannot be resected when the patients have highly advanced disease or when the primary sites are undefined. Such GEP-NETs cannot be evaluated with Ki-67 or the mitotic index. The aim of this study was to examine the prognosis of GEP-NETs that were ungraded by WHO G1-3 grading (U-NET group). METHODOLOGY: Between 2000 and 2011, 75 patients with sporadic GEP-NETs were treated at our institution. The prognosis of patients graded as new WHO grading (G-NET group) was compared with that of the U-NET group. Cox proportional hazard regression analyses were performed to estimate the risk factors for overall survival (OS). RESULTS: Overall 1-, 3- and 5-year survival rates were 90.7%, 79.9% and 74.9%, respectively. The odds ratio (OR) of patients with synchronous liver metastasis and U-NET was 1.73 (p=0.01) and 5.84 (p=0.002), respectively. Multivariate analyses of OS according to baseline characteristics revealed the only independent risk factor to be U-NET (OR, 3.95; p=0.02). CONCLUSIONS: The malignant potential of U-NET may be no less than that of G-NET, while WHO-G3 patients have the worst prognoses in the G-NET group.

Comparing of IRS and Her2 as immunohistochemical scoring schemes in gastroenteropancreatic neuroendocrine tumors.

UNLABELLED: Neuroendocrine tumors (NET) are known for an overexpression of somatostatin receptors (SSTR). In light of very few and partially contradictory publications, the present study aims to achieve a definite immunohistochemical (IHC) quantification and assessment of the distribution of all five SSTR-subtypes on NET and to evaluate an implementable scoring system, comparing the immunoreactive score of Remmele and Stegner (IRS) to the Her2-score. In 21 patients 40 different tumor tissues were IHC analysed using polyclonal antibodies for SSTR1 and 3-5 and the monoclonal antibody UMB-1 for SSTR2A. SSTR expression was quantitatively evaluated according to HER2-score and IRS, correlated among each other and to the maximum standardized uptake value (SUVmax) in tumor lesions as measured by PET/CT using 68Ga-DOTA-NOC. RESULTS: According to the IRS, the expression of SSTR2A and 3 predominated equally with 84%, followed by SSTR4 (44%) and SSTR1 and 5 (32%). With the Her2-scoring system the most frequent subtype was found to be SSTR2A (68%), followed by SSTR3 (64%), SSTR1 (44%), SSTR5 (40%), and SSTR4 (36%). The IRS-classification and the Her2-score were found to be statistically comparable, and their correlation is highly significant for each SSTR assessment (p<0.01). CONCLUSION: The results of the analyses revealed heterogeneous expression patterns. SSTR2A and 3 were highly expressed, demonstrating the importance of SSTR for diagnostics and therapy. Relatively high frequency of SSTR3 and 4 on NET give reasons to try pansomatostatin analogues for therapy rather than concentrating only on the SSTR2A. Statistically, none of the immunohistochemical scores was superior. However, due the heterogeneity of the cytoplasmic staining justice we propose the IRS as a uniform scoring scheme for IHC NET diagnostic.

A step further in the analysis of human bile proteome.

Bile was shown to collect proteins known as potential cancer biomarkers. Thorough proteomic analysis of bile is of particular interest to search for new, more sensitive and more specific, biomarkers of cancers affecting the biliary tract and surrounding organs, such as the pancreas and the liver. Therefore, extending the knowledge of the bile proteome is highly relevant, but this has proved technically difficult. In this study, we describe a strategy that circumvents problems related to the biochemical complexity of this sample and the presence of high concentrations of interfering substances. Bile collected from a patient suffering from a biliary stenosis caused by a pancreatic adenocarcinoma was fractionated by a differential centrifugation scheme, involving a stepwise increase in centrifugation speeds. Pellets and the final supernatant were further fractionated by polyacrylamide gel electrophoresis and proteins were in-gel digested prior to LC-MS/MS analysis. This approach allowed the identification of 445 unique proteins with at least two peptides (812 proteins if single-hit proteins were included), which represents a 3-fold increase in the knowledge of bile proteome. The subsequent literature comparison revealed that numerous biliary proteins identified in this sample were related to pancreas cancer. Immunoblot analysis of some known tumor markers revealed that they were preferentially associated with the soluble fraction rather than with pellets containing cellular components.