HHLA2 is expressed in pancreatic and ampullary cancers and increased expression is associated with better post-surgical prognosis.

BACKGROUND: HHLA2 is a recently discovered member of the B7-family of immune checkpoint molecules with limited expression in normal tissues but overexpression in several types of cancer. The aim was to determine the expression, prevalence and biological relevance of HHLA2 protein expression in two closely related human cancer types, namely pancreatic cancer and ampullary cancer. METHODS: HHLA2 expression levels were retrospectively determined by immunohistochemistry in tissue micro-arrays of surgically resected tumours of 122 pancreatic cancer patients and 72 patients with ampullary cancer of the pancreato-biliary subtype. RESULTS: HHLA2 was expressed at variable levels by tumour cells in 67% of pancreatic tumours and 93% of ampullary tumours. In the combined cohort high tumoural HHLA2 expression levels were significantly associated with delayed cancer recurrence and improved post-operative cancer-specific survival. The association of HHLA2 expression with cancer-specific survival and recurrence was statistically significant for the pancreatic cancer subgroup while a similar trend was found for the ampullary cancer subgroup. In multivariable analysis together with clinicopathologic characteristics, higher HHLA2 expression was an independent predictor of cancer-specific survival. CONCLUSION: The wide expression of HHLA2 in tumour cells and its association with cancer recurrence and patient survival suggest that HHLA2 represents a relevant immune checkpoint molecule in pancreatic and ampullary cancers.

[Combined application of immunohistochemical markers to identify pathologic subtypes of ampullary carcinoma and its clinical significance].

Objective: To investigate the expression of immunomarkers CK7, CK20, CK17, CDX2, MUC1 and MUC2 in primary adenocarcinoma of the ampulla of Vater, to explore the role of these markers in the histopathologic subclassification of ampullary carcinoma; and to provide biologic basis for precision treatment of patients with different types of ampullary carcinoma. Methods: Forty-two cases of primary ampullary carcinoma were collected at Peking University People's Hospital, from 2012 to 2018 year. There were 22 males and 20 females. Aged range 42 to 88 years old, with mean aged (62±11) years. Among the patients, 6 was high differentiation, 19 median differentiation, and 17 low differentiation. Immunohistochemical studies on the expression of CK7, CK20, CK17, CDX2, MUC1 and MUC2 were performed in 42 cases of primary ampullary carcinoma. The relationship between different ampullary carcinoma subtypes and clinicopathologic survival data was analyzed using SPSS 16.0 statistical software. Results: Three histopathologic subtypes were observed. Among 42 cases, 8(19.0%)were classified as intestinal subtype, which showed a positive expression rate of 8/8 for both CK20 and CDX2, and 5/8 for MUC2. Both CK7 and CK17 were weakly expressed in one case (1/8). No expression was observed for MUC1 in this subtype. Twenty-two (52.4%,22/42) cases were classified as pancreaticobiliary subtype, which showed a positive expression rate of 100.0%(22/22) for both CK7 and MUC1, and 90.9% (20/22) for CK17. No expression was observed for CK20, CDX2 and MUC2.The remaining 12 (28.6%) cases were classified as mixed subtype, which showed variable expression patterns. The expression frequencies of these 6 immunomarkers in different subtypes of ampullary carcinoma did not correlate with various clinicopathologic factors such as patient gender and age, tumor size, histologic differentiation, pancreatic and bile duct invasion, or the depth of duodenal invasion. However, stage Ⅲ+Ⅳ diseases were more commonly seen in pancreaticobiliary type (63.6%,14/22) than intestinal type (2/8) and mixed type (3/9; χ(2)=6.508, P=0.039). Follow-up data showed a trend of better survival rate for patients with intestinal subtype than those with mixed and pancreaticobiliary subtypes. Conclusions: Ampullary carcinoma can be subclassified into three different subtypes using a panel of six immunomarkers, especially for the identification of subtypes of poorly differentiated carcinoma. CK7, CK17 and MUC1 are major markers of pancreaticobiliary subtype, whereas CK20, CDX2 and MUC2 are useful markers for intestinal subtype. The mixed subtype variably expresses these markers. The prognosis of patients with intestinal subtype appears better than that of pancreaticobiliary and mixed subtypes. Accurate subtyping of ampullary carcinoma is clinically important to patient management and prognosis assessment.

Sequencing of 279 cancer genes in ampullary carcinoma reveals trends relating to histologic subtypes and frequent amplification and overexpression of ERBB2 (HER2).

The biological relevance of histological subtyping of ampullary carcinoma into intestinal vs pancreaticobiliary types remains to be determined. In an effort to molecularly profile these subtypes of ampullary carcinomas, we conducted a two-phase study. In the discovery phase, we identified 18 pancreatobiliary-type ampullary carcinomas and 14 intestinal-type ampullary carcinomas using stringent pathologic criteria and performed next-generation sequencing targeting 279 cancer-associated genes on these tumors. Although the results showed overlapping of genomic alterations between the two subtypes, trends including more frequent KRAS alterations in pancreatobiliary-type ampullary carcinoma (61 vs 29%) and more frequent mutations in APC in intestinal-type ampullary carcinoma (43 vs 17%) were observed. Of the entire cohort of 32 tumors, the most frequently mutated gene was TP53 (n=17); the most frequently amplified gene was ERBB2 (n=5); and the most frequently deleted gene was CDKN2A (n=6). In the second phase of the study, we aimed at validating our observation on ERBB2 and assessed ERBB2 amplification and protein overexpression in a series of 100 ampullary carcinomas. We found that (1) gene amplification and immunohistochemical overexpression of ERBB2 occurred in 13% of all ampullary carcinomas, therefore providing a potential target for anti-HER2 therapy in these tumors; (2) amplification and immunohistochemical expression correlated in all cases, thus indicating that immunohistochemistry could be used to screen tumors; and (3) none of the 14 ERBB2-amplified tumors harbored any downstream driver mutations in KRAS/NRAS, whereas 56% of the cases negative for ERBB2 amplification did, an observation clinically pertinent as downstream mutations may cause primary resistance to inhibition of EGFR family members.

MicroRNA profiling in periampullary carcinoma.

BACKGROUND: MicroRNA expression patterns in many physiological and oncogenic processes have been established. However, the role of aberrant miRNA expression in periampullary carcinoma (PAC) has not been elucidated. We hypothesize that PAC may have differential expression of miRNAs which may differentiate the tumor histological subtypes. METHODS: Fresh paired tumor and control samples were collected from the PAC patients undergoing Whipple's pancreaticoduodenectomy. Microarray miRNA profiling was performed utilizing tumor (n = 40) and control tissues; adjacent normal pancreas (n = 22), six each distal CBD, duodenum and ampulla. Data obtained was subjected to statistical and bioinformatic analysis. Differentially expressed miRNAs obtained were validated using qPCR in an independent set of samples. RESULTS: Comparison of PAC tissue samples with controls revealed 29 common and differentially expressed miRNAs (20 upregulated and 9 downregulated) with a higher statistical significance (p < 0.001) and fold change (log2 FC > 1.5). A subset of 16 miRNAs (15 overexpressed and 1 underexpressed) differed in expression levels between pancreatobiliary and intestinal subtypes. Among these, miR-375, miR-31 and miR-196a expressions varied significantly between histological subtypes. Differential expression profiles of miRNAs specific to TNM staging was also observed in PAC subtypes. Target gene prediction for the differentially expressed miRNAs in PAC revealed that target genes are enriched for certain pathways. Particularly, Wnt signaling pathway genes appear to be relevant targets for most of the differentially expressed miRNAs. CONCLUSION: Differentially expressed common miRNA signatures identified in PAC subgroups may have a role in pathogenesis of PAC and miR-375, miR-31 and miR-196a expression patterns may differentiate PAC subtypes.

A rare case of neuroendocrine cell tumor mixed with a mucinous component in the ampulla of Vater.

BACKGROUND: A rare case of neuroendocrine cell tumor (NET) having both conventional and mucinous components was reported. Mucinous NET is rarely encountered in the pathological diagnosis of gastrointestinal (GI) tumors. Here we examined the mechanism for transformation of conventional NETs into mucinous NETs. CASE PRESENTATION: Macroscopic examination revealed a tumor with ulceration in the ampulla of Vater that measured 1.7 cm in its largest diameter. Histologically, the tumor comprised two components: a tubular/ribbon-like feature and small nests floating in a mucinous lake. The tumor nests showed sheet, nest and ribbon-like structures of small cells having eosinophilic cytoplasm as well as small-sized nuclei with dense hyperchromatin. Immunohistochemical analysis showed tumor cells positive for pan-endocrine markers (synaptophysin, CD56, INSM1 and chromogranin). Based on the histological findings, the solid and mucinous components were diagnosed as conventional and mucinous NETs, respectively. Grading was NET G2 based on 12.8% and 13.2% Ki-67-positive cells in the solid and mucinous components, respectively. Immunohistochemically, the mucin phenotype of this tumor was gastric and intestinal. Only the mucinous NET component had cytoplasmic CD10 expression. Examination using a customized gene panel detected only a DPC4 mutation, which was limited to the mucinous component. CONCLUSIONS: Coexistence of conventional and mucinous NETs could provide important insight into evaluating the NET subtype histogenesis. Moreover, molecular alterations including cytoplasmic expression of CD10 and the DPC4 mutation can contribute to interpretation of tumor pathogenesis.

Loss of E-cadherin and beta-catenin is correlated with poor prognosis of ampullary neoplasms.

BACKGROUND AND OBJECTIVES: Distant metastasis resulting from carcinoma cell detachment from the primary tumor involves modification of adhesion molecules. This study was conducted to examine the correlation of E-cadherin/beta-catenin expression with survival and recurrence in ampullary neoplasms. METHODS: Patients with diagnoses of ampullary neoplasms were enrolled in the study. Demographics, operative findings, and histopathological data were collected by retrospective chart review. Expression of E-cadherin and beta-catenin were detected by immunohistochemistry. RESULTS: A total of 110 patients were enrolled in the study. Preservation of membranous staining of E-cadherin was noted in 41 (37%) patients, aberrant cytoplasmic staining in 48 (44%) patients, and complete loss in 21 (19%) patients. Loss of E-cadherin was associated with pancreatic invasion, recurrence, and poor prognosis. Membranous staining of beta-catenin was noted in 65 (59%) patients, cytoplasmic or nuclear accumulation in 16 (15%) patients, and complete loss in 29 (26%) patients. Loss of beta-catenin expression was associated with tumor markers, ulcerative type, liver metastases, and poor prognosis. Pancreatic invasion, lymph node involvement, and loss of beta-catenin expression were predictors of disease recurrence. CONCLUSIONS: Loss of the E-cadherin/beta-catenin complex is related to poor prognosis in ampullary cancer. Loss of beta-catenin is predictor of recurrence in multivariate analysis.

A case of gangliocytic paraganglioma in the ampulla of Vater.

BACKGROUND: Duodenal gangliocytic paraganglioma is an extremely rare tumor and few cases have been reported to date. CASE PRESENTATION: The authors report a case of gangliocytic paraganglioma verified by post-op pathology after pancreaticoduodenectomy for a tumor in the ampulla of Vater. The 56-year-old male patient concerned visited our emergency room with melena that started one week prior to hospitalization. The patient was diagnosed to have a tumor in the ampulla of Vater with bleeding on its surface. However post-op, he was diagnosed as having gangliocytic paraganglioma by immunohistochemistry. CONCLUSION: This tumor has precise clinical implications, and if continuous follow up is conducted after careful diagnosis and surgical treatment, invasive major operations, such as, radical pancreaticoduodenectomy can be avoided.

Expression of CD24, P-cadherin and S100A4 in tumors of the ampulla of Vater.

Carcinomas of the Vaterian system are rare and presumably arise from preexisting adenomas (adenoma-carcinoma-sequence). Usually, biopsies are obtained to confirm and specify endoscopic findings, but differentiating reactive atypia from dysplasia or dysplasia from invasive carcinoma can sometimes be difficult or even impossible on morphological criteria alone. In case of invasive carcinoma, furthermore, the precise classification of carcinoma subtypes needs to be established since the distinct subtypes differ significantly in terms of clinical outcome. The cell adhesion proteins CD24, P-cadherin and S100A4 were shown to be expressed in several carcinomas and in dysplastic epithelium but only rarely in normal mucosa. We therefore investigated their expression in 177 carcinoma, 114 adenoma and 152 normal mucosa specimens of the ampulla of Vater. Although the expression of the cell adhesion proteins did not differ between the carcinoma subtypes, marked differences between normal mucosa, adenoma and carcinoma samples were observed. All marker proteins were expressed in less than 7% of normal mucosa samples (S100A4 in only 1% of cases) and showed an increasing expression from adenoma to invasive carcinoma. Our findings suggest that P-cadherin and S100A4 are helpful in discriminating normal mucosa or reactive atypia from neoplastic lesions. CD24 and S100A4, furthermore, can assist in the differential diagnosis of dysplasia vs invasive carcinoma.

Human equilibrative nucleoside transporter 1 (hENT1) protein is associated with short survival in resected ampullary cancer.

BACKGROUND: Gemcitabine is an acceptable alternative to best supportive care in the treatment of advanced biliary tract cancers. The human equilibrative nucleoside transporter 1 (hENT1) is a ubiquitous protein and is the major means by which gemcitabine enters human cells. Moreover, recent reports indicate a significant correlation between immunohistochemical variations of hENT1 in tumor samples and survival after gemcitabine therapy in patients with solid tumors. MATERIALS AND METHODS: We used immunohistochemistry to assess the abundance and distribution of hENT1 in tumor samples from radically resected cancer of the ampulla, and sought correlations between immunohistochemical results and clinical parameters including disease outcomes. RESULTS: In the 41 individual tumors studied, 12 (29.3%) had uniformly high hENT1 immunostaining. Statistical analysis showed a significant correlation between hENT1 and Ki-67 (P = 0.04). No statistical significant differences were found between immunohistochemical findings and patient characteristics (sex, age, and tumor-node-metastasis). On univariate analysis, hENT1 and Ki-67 expression were associated with overall survival (OS). Specifically, those patients with overexpression of hENT1 showed a shorter OS (P = 0.022) and those with high Ki-67 staining showed a shorter survival (P = 0.05). CONCLUSIONS: hENT1 expression is a molecular prognostic marker for patients with resected ampullary cancer and holds promise as a predictive factor to assist in chemotherapy decisions.

Significance of GLUT1 expression in adenocarcinoma and adenoma of the ampulla of Vater.

The expression of glucose transporter protein 1 (GLUT1) in malignant tumors is increased due to the higher metabolic needs of the proliferating cell populations. Aberrant GLUT1 expression is exhibited in a wide spectrum of epithelial malignancies and their precursors, which occur with low frequency and intensity; aberrant GLUT1 expression does not occur in normal epithelial cells. The expression of GLUT1 in tumors of the ampulla of Vater was evaluated on immunohistochemistry, and the relationships of GLUT1 expression to histological parameters and p53 expression were analyzed. Twenty-one (58.3%) of 36 adenocarcinomas and three (17.6%) of 17 adenomas had GLUT1 immunoreactivity. None of the regenerating or normal epithelia had any immunoreactivity. No significant relationships were found between GLUT1 expression and histological parameters or p53 expression. It was found that histological subtypes originated from different epithelium were strongly related to different macroscopic types. In the ampulla of Vater, GLUT1 expression was associated with malignant change, and might be a useful marker of malignancy.

Spindle and giant cell type undifferentiated carcinoma arising in the common bile duct: a case report.

We report on a 61-year-old Japanese male with a pedunculated tumor in the common bile duct. The tumor consisted of two types of neoplastic cells. The majority showed atypical spindle- and giant-shaped features and proliferated densely in an inflammatory stroma, revealing a sarcomatous pattern. They expressed vimentin, KL-1, and CAM5.2. The remaining minority showed glandular and tubular features, occupied only less than 5%, located only in the tumor surface, and expressed wide spectrum keratin, KL-1, CAM5.2, epithelial membrane antigen, AE1/AE3, and carcinoembryonic antigen. CD68-positive osteoclast-like giant cells were also observed. Therefore, the patient was diagnosed as having an undifferentiated carcinoma, spindle and giant cell type.

Synchronous GIST with osteoclast-like giant cells and a well-differentiated neuroendocrine tumor in Ampula Vateri: coexistence of two extremely rare entities.

Mesenchymal tumors of the gastrointestinal system with variable histopathological appearances and constant expression of CD117 are known as gastrointestinal stromal tumors (GISTs). Neuroendocrine tumors may be seen in the gastrointestinal system and other organ systems of the body. We report a 44-year-old male patient with a 6.5 x 3 x 6cm mass located in the Ampulla of Vater. Histopathologic examination revealed a GIST with a marked nuclear pleomorphism and a high mitotic rate, and that was rich in osteoclast-like giant cells (OGC). Immunohistochemically, GIST was positive for CD117, while OGCs were negative for CD117 and positive for CD68 and alpha1-antitrypsin. There was also found a well-differentiated neuroendocrine tumor near the GIST, in the serosal aspect of the duodenum at the point of the Ampulla of Vater. This second tumor was 20mm in diameter, and was relatively well circumscribed with few glands invading the GIST. This tumor was positive for synaptophysin and chromogranin. Neither mitosis nor vascular invasion was observed. The patient had no familial history or clinical manifestations of neurofibromatosis. This case presents the unique synchronous existence of two extremely rare entities, a GIST with OGC and a well-differentiated neuroendocrine tumor, both located in the Ampulla of Vater.

Prognostic value of mucins in the classification of ampullary carcinomas.

The ampulla of Vater is of high clinical relevance with regard to influx of chyme, ascending inflammation, intubation during diagnostic and therapeutic endoscopic investigation, therapeutic papillotomy, and especially to malignant transformation. Little is known about the distribution of mucins in the ampulla. In this study, we have investigated the mucin distribution in the normal ampulla of Vater and compared it to duodenal mucosa and Brunner glands. Expression of mucins in the ampulla of Vater and duodenum was monitored by reverse transcription-polymerase chain reaction and localization of the products by immunohistochemistry. The samples investigated originated from 30 autopsy cases. Mucins MUC1, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7, and MUC8 were expressed in the ampulla of Vater. Immunohistochemistry revealed production of MUC4, MUC5AC, MUC5B, and MUC6. The mucin composition varied in comparison with the duodenum referring to MUC2, MUC7, and MUC8. Detected mucins contribute to innate immunity, epithelial restitution, and protection against the aggressive secretions of the liver, gall bladder, and pancreas. By cross-linking, they influence the rheological properties of the secretions in the ampulla and facilitate unidirectional flow into the duodenum. Knowledge of their pattern of expression has prognostic value with regard to the detection of malignancy. The observed differences in the mucin distribution between the duodenum and the ampulla of Vater support the use of MUC2, MUC7, and MUC8 as useful tool in the classification of ampullary carcinomas.

Ampullary neuroendocrine tumor diagnosed by endoscopic papillectomy in previously confirmed ampullary adenoma.

Ampullary adenoma is a common indication for endoscopic papillectomy. Ampullary neuroendocrine tumor (NET) is a rare disease for which complete surgical resection is the treatment of choice. However, because of the morbidity and mortality associated with surgical resection, endoscopic papillectomy is increasingly used in selected cases of low grade, with no metastasis and no invasion of the pancreatic or bile duct. Also, confirmed and complete endoscopic resection of ampullary NET accompanied by adenoma has not been reported to date. We report herein a rare case of an ampullary NET accompanied with adenoma, which was successfully and completely resected via endoscopic papillectomy. Prior to papillectomy, this case was diagnosed as an ampullary adenoma.

High-grade neuroendocrine carcinoma of the ampulla of vater: a clinicopathologic and immunohistochemical analysis of 14 cases.

We describe the clinical and pathologic features of 14 cases of high-grade neuroendocrine carcinoma (HGNEC) of the ampulla of Vater classified according to WHO classification of lung tumors into small cell carcinoma (SCC, 6 cases) and large cell neuroendocrine carcinoma (LCNEC, 8 cases) types. The immunohistochemical findings were compared with those of 13 cases of primary poorly differentiated ampullary adenocarcinomas (PDACA) lacking neuroendocrine morphology. The mean age of 10 males and 4 females was 70 years. The mean tumor size was 2.5 cm. Ten of 13 patients had lymph node metastases (mean, 2.3 nodes involved). Documented sites of distant metastases included brain and liver. Overall, 64% of patients with ampullary HGNEC died of disease (mean follow-up, 14.5 months). Four patients had no evidence of disease after resection (mean, 20 months). Half of the tumors were associated with adenomas of the adjacent mucosa, 2 with high-grade dysplasia. Two HGNECs were combined with a conventional adenocarcinoma and another with a squamous cell carcinoma component. By immunohistochemistry, the HGNECs were positive for cytokeratins (AE1/AE3, 100%; Cam5.2, 67%; CK7, 87%; CK20, 38%), similar to the pattern found in PDACAs. p27 expression was lost in 1 case of HGNEC and in all PDACAs. Retinoblastoma (Rb) protein expression was lost in 60% of HGNECs and in none of the PDACA cases. In conclusion, HGNECs of the ampulla are rare (2%-3% of ampullary tumors in our material). The clinical course parallels that of their pulmonary counterparts and appears to be worse than that of locally advanced ampullary adenocarcinomas. The association with adenoma and or conventional adenocarcinoma components may suggest a common pathway in the initial carcinogenesis of these two types of tumors. Loss of Rb expression, a characteristic finding in pulmonary SCCs, is present in almost half of ampullary HGNECs. In contrast, p27 expression is lost in PDACAs and retained in most HGNECs. Thus, there are differences in the molecular phenotypes of these two types of ampullary carcinoma, supporting the distinction of poorly differentiated carcinomas with a neuroendocrine phenotype from those without.

Prognostic value of Bax, Bcl-2, p53, and TUNEL staining in patients with radically resected ampullary carcinoma.

BACKGROUND: There is a lack of data in the literature concerning the identification of potential prognostic factors in ampullary adenocarcinoma. AIMS: To examine the prognostic significance of Bax, Bcl-2, and p53 protein expression and the apoptotic index in a large cohort of uniformly treated patients with radically resected ampullary cancer. METHODS: All patients with a pathological diagnosis of ampullary cancer and radical resection were evaluated. Expression analysis for p53, Bax, and Bcl-2 was performed by immunohistochemistry. Apoptotic cells were identified by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). RESULTS: Thirty nine tumour specimens from patients with radically resected ampullary adenocarcinoma were studied. A positive significant correlation between Bax and p53 expression was found by rank correlation matrix (p < 0.001). A trend towards a positive correlation was found between the apoptotic index and p53 expression (p = 0.059). By univariate analysis, overall survival was influenced by Bax expression, p53 expression, and TUNEL staining (p = 0.001, p = 0.01, and p = 0.03, respectively). Bcl-2 expression did not influence overall survival in these patients (p = 0.55). By multivariate Cox regression analysis, the only immunohistochemical parameter that influenced overall survival was Bax expression (p = 0.020). CONCLUSIONS: These results provide evidence that apoptosis may be an important prognostic factor in patients with radically resected ampullary cancer. This study is the first to assess the clinical usefulness of Bax expression in radically resected ampullary cancer.

Amphicrine carcinoma of the ampullary region.

Amphicrine carcinoma is a peculiar tumor in which the cells have both exocrine and neuroendocrine differentiation, with mucus and neuroendocrine granules within the cytoplasm. In the 2010 WHO classification of tumors of the digestive tract, they have been included in the intermediate-grade malignant category of mixed adenoneuroendocrine carcinomas (MANECs). These tumors are extremely rare in the gastrointestinal tract. Four cases have been reported in the stomach, three in the pancreas, and one in the liver. To the best of our knowledge, this report describes the first case of amphicrine carcinoma in the ampullary region.

Mixed adenoneuroendocrine carcinoma of the ampulla: two case reports.

Mixed adenoneuroendocrine carcinoma (MANEC) is a malignant tumor with adenocarcinoma and neuroendocrine components, with ≥30% of each component required. MANEC of the ampulla is rare. To the best of our knowledge, only 15 cases of MANEC of the ampulla have been reported in the English-language literature. Here, we report two cases of MANEC of the ampulla in two women aged 43 and 60 years, which was confirmed by histology after pancreaticoduodenectomy. These tumors contained neuroendocrine and adenocarcinoma components. The neuroendocrine components were positive for chromogranin A (CgA), synaptophysin (Syn) and CD56 by immunostaining. The adenocarcinoma components were negative for CgA, Syn and CD56. Both cases were T3N0M0 (Stage IIIA). They survived for 15 and 20 mo after surgery, respectively. A brief discussion about the histopathological features, clinical behavior and treatment of MANEC of ampulla, and review of the relevant literature are presented.