Expression of cartilage oligomeric matrix protein in periampullary adenocarcinoma is associated with pancreatobiliary-type morphology, higher levels of fibrosis and immune cell exclusion.

Cartilage oligomeric matrix protein (COMP) is an emerging regulator of tumor progression. The aim of this study was to evaluate the expression of COMP in periampullary adenocarcinoma with respect to prognostic value for survival and relapse, levels of fibrosis and infiltrating immune cells. COMP expression was evaluated using immunohistochemistry in primary tumors and subsets of paired lymph node metastases in tissue microarrays including 175 patients with periampullary adenocarcinoma. Collagen content was assessed with Sirius Red-Fast Green staining. High COMP levels were detected in cancer cells and in stroma, in 46% and 57% of the patients, respectively. High COMP expression was strongly associated with more aggressive pancreatobiliary-type (PB-type) compared to intestinal-type tumors (p < .0001). Importantly, high expression of COMP correlated with the exclusion of cytotoxic T-cells from the cancer cell compartment of the tumors, particularly in PB-type tumors. Higher levels of fibrosis measured by the density of collagen fibers correlated with high COMP levels in both cancer cells and stroma. This in turn could lead to exclusion of cytotoxic T-cells from accessing the cancer cells, a recognized immunotherapy resistance mechanism. Targeting COMP could therefore be considered as a novel therapeutic strategy in PB-type periampullary adenocarcinoma.

Identification of ampullary carcinoma mixed subtype using a panel of six antibodies and its clinical significance.

OBJECTIVES: To investigate the function of immunomarkers CK7, CK20, CK17, CDX2, MUC1, and MUC2 in the identification of primary ampullary carcinoma mixed subtype. METHODS: Forty-two cases of primary ampullary carcinoma were performed by immunohistochemical studies. The correlation between the mixed subtype and the other two subtypes and patient survival data was analyzed using the SPSS 16.0 statistical software. RESULTS: Among 42 cases, 12 (28.6%) cases were classified as mixed subtype, which showed variable expression patterns: 91.7% (11/12) for CK7, 83.3% (10/12) for CK20; 66.7% (8/12) for CK17, CDX2, and MUC1; and 50% (6/12) for MUC2. Ten (83.3%) mixed types coexpressed four or more immunomarkers. Eight (19%) intestinal subtypes mainly showed a positive expression of CK20, CDX2, and MUC2. Twenty-two (52.4%) pancreaticobiliary subtypes showed a positive expression of CK7, MUC1, and CK17. Stages III and IV diseases in mixed subtype (25%) and intestinal subtype (25%) were less than pancreaticobiliary subtype(63.6%) (p = 0.039). Follow-up data appeared to show a better survival rate for patients with mixed subtype than those with pancreaticobiliary subtypes. CONCLUSION: Immunohistochemical staining provided a more reliable means of diagnosing mixed ampulla carcinoma. Accurate subtyping of ampullary carcinoma is clinically important to select effective chemotherapy regimens and to assess disease prognosis.

PD-L1 overexpression in ampulla of Vater carcinoma and its pre-invasive lesions.

AIMS: PD-1/PD-L1 checkpoint immunotherapy has been proposed recently as a promising treatment in relapsed/refractory disease, used eventually in combination with traditional chemotherapy in different cancer settings. To date, no data are available concerning PD-L1 expression in ampulla of Vater carcinoma and its pre-invasive lesions. METHODS AND RESULTS: We assessed the immunohistochemical expression of PD-L1 in a series of 26 ampullary adenocarcinomas, 50 ampullary dysplastic lesions and 10 normal duodenal mucosa samples. Moreover, in all cases DNA mismatch repair proteins status was investigated. PD-L1 was expressed in seven of 26 (26.9%) invasive carcinomas and three of 50 (6.0%) dysplastic samples. Most of the PD-L1-positive tumours (seven of 10) were intestinal-type and poorly differentiated (G3). The number of PD-L1-positive stromal lymphoid cells was significantly higher in dysplastic and invasive lesions than in the normal samples (P = 0.011). Nineteen dysplastic lesions and eight invasive carcinomas did not show any evident epithelial or stromal PD-L1 expression. Four of the carcinomas were mismatch repair-deficient and two of these were PD-L1-positive. Furthermore, mismatch repair-deficient lesions showed a significantly higher average of PD-L1-positive stromal lymphoid cells than those of neoplastic PD-L1-negative samples (62.8 versus 21.6; P < 0.001). CONCLUSIONS: The present results suggest a role of the PD-1/PD-L1 axis in ampullary adenocarcinomas, and therefore this may also prompt consideration of checkpoint immunotherapy as a novel promising treatment for these tumours.

Tumor cell expression of immune inhibitory molecules and tumor-infiltrating lymphocyte count predict cancer-specific survival in pancreatic and ampullary cancer.

Understanding the mechanisms of immune resistance in pancreatic and ampullary cancers is crucial for the development of suitable biomarkers and effective immunotherapeutics. Our aim was to examine the expression of the immune inhibiting molecules PD-L1, Galectin-9, HVEM, IDO and HLA-G, as well as CD8+ and FoxP3+ tumor infiltrating lymphocytes (TIL), in pancreatic and ampullary cancers, and to relate their individual, as well as their combined expression, to cancer survival. Tumor tissue from 224 patients with resected pancreatic (n = 148) and ampullary (n = 76) cancer was used to construct tissue-microarrays. Expression of immune inhibitory molecules and TIL was examined by immunohistochemistry. We show that immune inhibitory molecules are prevalently expressed. Moreover, high tumor expression of PD-L1 (p = 0.002), Gal-9 (p = 0.003), HVEM (p = 0.001), IDO (p = 0.049), HLA-G (p = 0.004) and high CD8/FoxP3 TIL ratio (p = 0.006) were associated with improved cancer-specific survival. All immune biomarkers, with the exception of IDO, were individually predictive of cancer-specific survival when adjusted for clinicopathologic characteristics. For every additional immune biomarker present survival was almost two-fold prolonged (HR 0.57 95%CI 0.47-0.69, p < 0.0001). When patients with pancreatic and ampullary cancer were analyzed separately the results were similar. We conclude that pancreas and ampullary cancers are rich in expression of immune-inhibitory molecules. These molecules can be targets for future immunotherapeutics, as well as form powerful immunological biomarkers. We propose that such immune biomarker panels be included in future prospective immunotherapy trials.

Survival Outcomes According to Adjuvant Treatment and Prognostic Factors Including Host Immune Markers in Patients with Curatively Resected Ampulla of Vater Cancer.

BACKGROUND: Ampulla of Vater cancer (AoV Ca) is a rare tumor, and its adjuvant treatment has not been established. The purpose of this study was to find out prognostic factors including host immunity and role of adjuvant treatment in AoV Ca. METHODS AND FINDINGS: We reviewed 227 AoV Ca patients with curative resection. Clinical characteristics, adjuvant treatment, disease-free survival (DFS) and overall survival (OS) were analyzed. Among all patients, 63.9, 36.1 and 33.9% had T1/T2, T3/T4 stage and lymph node-positive disease (LN+), respectively. OS of all patients was 90.9 months (95% CI: 52.9-129.0). OS was different according to neutrophil-to-lymphocyte ratio (HR 1.651, 95% CI: 1.11-2.47), platelet-to-lymphocyte ratio (HR 1.488, 95% CI: 1.00-2.21) and systemic inflammatory index (HR 1.669, 95% CI: 1.13-2.47). In multivariate analysis, adverse prognostic factors for OS included vascular invasion (HR 2.571, 95% CI: 1.20-5.53) and elevated CA 19-9 (HR 1.794, 95% CI: 1.07-3.05). A total of 104 patients (46.3%) received adjuvant treatment (25 out of 111of T1/T2 & LN (-), 79 out of 116 of T3/T4 or LN (+)). In T3/T4 or LN (+) stage, adjuvant CCRT with maintenance chemotherapy provided the longest OS (5-year OS rate: 47.0 vs. 41.4%). CONCLUSIONS: Vascular invasion and elevated CA 19-9 were adverse prognostic factors in resected AoV Ca. In T3/T4 or LN (+) stage, adjuvant CCRT with maintenance chemotherapy provided the best survival outcome. Adjuvant treatment should be further defined in AoV Ca, especially with poor prognostic factors.

Increased IgG4+ cells in duodenal biopsies are not specific for autoimmune pancreatitis.

Endoscopic ampullary biopsies showing increased immunoglobulin (Ig) G4+ plasma cells have been reported as an alternative to pancreatic biopsy in diagnosing autoimmune pancreatitis (AIP). This study assessed whether increased IgG4+ cells can be seen outside the context of AIP. Fifty-four cases (45 duodenal or ampullary biopsies, 9 ampullae from pancreatic resections) were selected, and all specimens were immunostained for IgG4 and IgG. Duodenal or ampullary biopsies containing normal duodenal mucosa (n = 6) and increased intraepithelial lymphocytes without villous blunting (n = 7) were negative for IgG4. Increased IgG4+ cells (>10 per high-power field) were found in 7 cases of 18 serologically confirmed celiac disease patients and in 3 of 14 patients with duodenitis or gastric heterotopias. Two of 6 ampullae from patients with pancreatic cancer showed increased IgG4+ cells. In summary, 12 of 51 patients without AIP had duodenal biopsies or ampullae showing increased IgG4+ plasma cells. The finding of increased IgG4+ cells in duodenal biopsies is not specific for AIP without the correct clinical context.

Carcinoma of the ampulla of Vater: morphological and immunophenotypical classification predicts overall survival.

OBJECTIVES: The objective of the study was to verify if histopathological differentiation of ampullary carcinoma after surgical resection may be related to survival. METHODS: The prognostic role of an accurate histological and immunohistochemical classification has been investigated in a multicentric series of carcinoma of the ampulla of Vater. Immunohistochemical expression of cytokeratin 7 (CK7) and CK20 were analyzed in the different morphological histotypes of ampullary cancers, and results were compared with overall survival. RESULTS: Of 72 ampullary cancers, 48.6% were classified as pancreaticobiliary-type carcinomas, 43.1% were classified as intestinal-type carcinomas, and 8.3% were classified as "unusual"-type carcinomas. Cytokeratin 20 was expressed in 28 (90.3%) of the 31 intestinal-type carcinomas, whereas it was always negative in the pancreaticobiliary histotype; CK7 was expressed in 32 (91.4%) of the 35 pancreaticobiliary-type carcinomas and in 18 (58.1%) of the 31 intestinal-type carcinomas. By univariate analysis, overall survival was influenced significantly by pathological T factor, lymph node involvement, and histological/immunohistochemical subtyping. Furthermore, using a multivariate Cox regression model, lymph node metastasis and CK20 were identified as significant independent factors related to prognosis. CONCLUSION: Our results prove the clinical use of ampullary cancer subclassification based on different histotypes and indicate the useful role of the CK7/CK20 expression profile for consistent histopathological classification and prognostic relevance.

IgG4 immunostaining of duodenal papillary biopsy specimens may be useful for supporting a diagnosis of autoimmune pancreatitis.

BACKGROUND: Autoimmune pancreatitis (AIP) is now considered to be part of an immunoglobulin G4 (IgG4)-related systemic fibroinflammatory disease. OBJECTIVE: We evaluated whether IgG4 immunostaining of duodenal papillary biopsy specimens is useful for supporting a diagnosis of AIP. DESIGN: A prospective study. SETTING: A tertiary academic center. PATIENTS/INTERVENTIONS: We obtained 2 forceps biopsy specimens from the major duodenal papilla (MDP) of 19 symptomatic AIP patients during ERCP before steroid administration. As a control, biopsy specimens were obtained from the MDP of patients with pancreatic cancer (n = 35), cholangiocarcinoma (n = 20), ampullary cancer (n = 11), ordinary chronic pancreatitis (n = 18), and AIP in remission (n = 16) and immunohistochemically examined. MAIN OUTCOME MEASUREMENTS: Specimens were considered positive for IgG4 immunostaining if there were more than 10 IgG4-positive plasma cells per high-power field. RESULTS: Positive IgG4 immunostaining of the MDP was found in 10 (53%) of 19 symptomatic AIP patients, but was absent in the control groups. Among symptomatic AIP patients (n = 19), 5 (83%) of 6 AIP patients with elevated serum IgG4 levels exhibited positive IgG4 staining of the MDP, whereas 5 (38%) of 13 AIP patients with normal serum IgG4 levels showed positive IgG4 staining of the MDP. Nine of 19 symptomatic AIP patients also underwent pancreatic biopsy, and positive IgG4 immunostaining of the MDP was observed in patients with positive pancreas IgG4 staining (4/6, 67%), but not in patients with negative pancreas IgG4 staining (0/3). LIMITATIONS: Small symptomatic AIP patient population. CONCLUSIONS: Positive IgG4 immunostaining of the MDP was an extremely specific and moderately sensitive tool for the diagnosis of AIP. IgG4 immunostaining of the MDP may be useful for supporting a diagnosis of AIP, especially when AIP is suspected clinically but serum IgG4 levels are normal or pancreatic tissue is not available.

IgG4+ to IgG+ plasma cells ratio of ampulla can help differentiate autoimmune pancreatitis from other "mass forming" pancreatic lesions.

Autoimmune pancreatitis (AIP) shows a unique spectrum of histologic features and commonly presents with an abundant IgG4-positive (IgG4+) plasma cell infiltration. However, differentiating AIP from other mass lesions, particularly pancreatic cancer [invasive ductal carcinoma (IDC)] can be clinically challenging. In this study, we evaluated the validity of IgG4 and IgG immunohistochemistry of ampullary and periampullary tissue for the diagnosis of AIP. Our study group consisted of 14 resected AIP cases with appropriate ampullary sections. Superficial ampullary tissue and "shouldering" duodenal mucosa were evaluated for several histologic variables. Immunohistochemistry for IgG4 and IgG was performed. The number of IgG4 and IgG-positive plasma cells was counted and an IgG4+ to IgG+ plasma cells ratio (IgG4/IgG ratio) was evaluated. A control cohort was composed of IDC (n=30) and chronic pancreatitis (CP) (n=29). Although an overlap was present between the groups, the overall inflammation and number of plasma cells in and around the ampulla was significantly increased in AIP compared with CP and IDC. Furthermore, although there was some overlap in the crude number of IgG4+ plasma cells of the ampullary and duodenal tissue between AIP, IDC, and CP, an IgG4/IgG ratio, especially of the ampulla, seems diagnostically useful in differentiating AIP from other "mass forming" lesions. When a cut-off of 0.10 was applied, the diagnostic sensitivity and specificity of the ampullary IgG4/IgG ratio was 86% and 95%, respectively. In conclusion, evaluation of ampullary histology and IgG4/IgG ratio might be proven beneficial in discriminating AIP from other mass forming pancreatic lesions.

Clinical significance of swollen duodenal papilla in autoimmune pancreatitis.

OBJECTIVES: To evaluate the clinical significance of a swollen main duodenal papilla and the associated immunohistopathologic findings in patients with autoimmune pancreatitis (AIP). METHODS: Seventeen consecutive patients with AIP registered between April 2001 and October 2005 who underwent both endoscopic retrograde cholangiopancreatography and endoscopic biopsy were enrolled in this study. The endoscopic features, stromal inflammatory cell infiltrate (SICI), and results of immunohistochemical examination of the duodenal papilla using IgG4, CD3, and CD79a antibodies were retrospectively reviewed. These findings in the AIP patients were compared with those in 12 patients with chronic alcoholic tumor-forming pancreatitis (CAP). The numbers of cells in the SICI and of IgG4-positive plasma cells per high-power field were counted in all the histopathologic specimens. RESULTS: A swollen main duodenal papilla was observed in 11 (11 [64.7%]/17) patients with AIP and 4 (4 [33.3%]/12) patients with CAP (P < 0.05). Resolution of the swollen main duodenal papilla was observed in all of these 11 patients with AIP (11 [100%]/11) in response to treatment with corticosteroids. On the other hand, the 6 patients without elevated serum IgG4 or a swollen duodenal papilla, but with a swollen pancreas, improved even without corticosteroid treatment. The number of cells in the SICI in the AIP patients was significantly higher than that in the CAP patients. Although in 13 of 17 AIP patients, infiltration by IgG4-positive plasma cells was detected in the duodenal papilla, no such significant infiltration of the duodenal papilla by IgG4-positive plasma cells was observed in the patients with CAP (P < 0.05). More predominant T-cell infiltration of the duodenal papilla was recognized in the AIP patients than in the CAP patients (P < 0.05). CONCLUSIONS: These results suggest that a swollen main duodenal papilla with IgG4-positive plasma cell and T-cell-dominant infiltration and an abundant stromal cell infiltrate are characteristic findings in AIP. We suggest that these findings may be valuable adjuncts to the diagnosis of AIP as well as for selecting suitable candidates for corticosteroid therapy.

Invasive micropapillary carcinomas of the ampullo-pancreatobiliary region and their association with tumor-infiltrating neutrophils.

Invasive micropapillary carcinoma, originally described as a distinctive type of invasive carcinoma in the breast, is being increasingly recognized as a separate entity in many other organs; however, it has not yet been documented in the pancreas or periampullary region. In this study, 313 pancreatic and 73 periampullary carcinomas were reviewed to investigate the micropapillary pattern in this location. Eight periampullary and eight pancreatic cases (4%) were composed at least focally (>20%) of invasive micropapillary carcinoma. The patients were 10 males and six females, mean age 69 years. The mean tumor size was 3.2 cm. Lymph node metastasis was detected in 11/15 cases. The median survival was 8 months (all were resected). Invasive micropapillary carcinoma was characterized by small, closely packed micropapillary clusters (without fibrovascular cores) lying within clefts. The cells had moderate degree of nuclear atypia. In nine cases, there was abundant inflammation composed of neutrophils concentrating around the tumor cells, both intraepithelial ('cannibalism') and stromal. Molecules implicated in abnormalities of tumor cell-stroma adhesion, galectin-3 and E-cadherin were expressed in the cytoplasm of 11/11 and 9/11 cases, respectively. Reversal of cell polarity was observed by MUC 1 in all 11 cases tested, which showed labeling in the stroma-facing surfaces of the micropapillary clusters, also confirming that the clefts are not merely a processing artifact, but indeed a true biologic alteration. In conclusion, invasive micropapillary carcinoma constitutes 4% of carcinomas in the pancreatic/periampullary region and is commonly associated with abundant neutrophilic infiltrates. Invasive miropapillary carcinoma appears to be more common in periampullary than in pancreatic invasive micropapillary carcinoma would qualify as poorly differentiated both based on pattern and the median survival (8 months)..

Small follicular lymphoma arising near the ampulla of vater: a distinct subtype of duodenal lymphoma?

A 49-yr-old Japanese woman underwent upper gastrointestinal endoscopy because of nonspecific dyspepsia. Endoscopy revealed a flat elevated lesion about 15 mm in diameter adjacent to the duodenal papilla, the surface of which was uneven and covered with whitish granules. Based on the results of histological examination with immunohistochemistry (positive for CD10, CD20, CD79a, and bcl-2 protein, negative for CD5 and cyclin D1), a diagnosis of grade 1/3 follicular lymphoma was established. Systemic staging examinations suggested the lymphoma was restricted to the mucosa and superficial portion of the submucosa in the duodenal wall. The patient was treated with a combination of CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisolone) and monoclonal anti-CD20 antibody (rituximab), in addition to radiotherapy. After six courses of this combination chemotherapy, complete regression of the lymphoma was observed. Although reports of small duodenal lymphoma (<20 mm or localized to the mucosa or submucosa) are extremely rare, the features of this case are characteristic of small duodenal lymphoma in terms of evolution around the ampulla of Vater, low-grade follicular type, occurrence in a women, occurrence in the fourth decade of life, and favorable outcome, and this type of tumor may need to be distinguished by pathogenesis and clinical behavior from various other gastrointestinal lymphomas.

Autoimmune pancreatitis associated with immune-mediated inflammation of the papilla of Vater: report on two cases.

Autoimmune pancreatitis (AIP) is defined histologically by periductal and interacinar lymphocytic infiltration. Immunohistochemically, the majority of these lymphocytes are identified as T cells. Epithelial HLA-DR antigen expression was also described as a marker of autoimmunity in this type of chronic pancreatitis. We report 2 cases, a 56-year-old man and a 29-year-old woman, with AIP associated with immune-mediated inflammation of the main duodenal papilla (MDP). Serologically, antinuclear antibody positivity was detected in the male patient. The female patient, treated medically for ulcerative proctitis, had no serological evidence of autoimmune disease. Macroscopic papillitis was present only in the male patient, and endoscopic biopsy samples were taken from this swollen MDP. Since we could not exclude malignancy, a pancreatic head resection was performed in both patients. The histologic and immunohistochemical studies of the resected specimens showed periductal T-lymphocytic infiltration in the pancreatic and papillary tissues. Furthermore, HLA-DR-antigen expression was also demonstrated in epithelial cells of the pancreas and MDP. The immunohistological features of endoscopic biopsy samples from the swollen MDP were identical as in the surgically resected specimens. Immune-mediated inflammation of the MDP may be associated with AIP.

The significance of proliferating cell nuclear antigen (PCNA) expression in cancer of the ampulla of vater in terms of prognosis.

Seventeen patients with cancer of the ampulla of Vater were studied retrospectively using immunohistochemical staining with a monoclonal antibody to the proliferating cell nuclear antigen (PCNA). The relationships between the PCNA-positive rate, being the number of PCNA-positive cancer cells to total cancer cells, the clinicopathological findings, and the clinical course were evaluated. The PCNA-positive rate in patients with lymph node metastasis (47%) was significantly higher than that in patients without metastasis (29%), while that in patients with advanced cancer invading the pancreatic parenchyma (47%), was significantly higher than that in patients with early cancer without invasion of the sphincter of Oddi (32%). All of five patients with early cancer are still alive, whereas five with semi-advanced cancer invading the sphincter of Oddi but not the pancreatic parenchyma, and two with a PCNA-positive rate of over 40% died of recurrent cancer. Of seven patients with advanced cancer, only one with a low PCNA-positive rate of 23% is alive, but the other six with a PCNA-positive rate of over 40% all died. The results suggest that the PCNA-positive rate provides a prognostic index for cancer of the ampulla of Vater.

Carcinoembryonic antigen (CEA) in benign and malignant epithelium of the gall bladder, extrahepatic bile ducts, and ampulla of Vater.

Carcinoembryonic antigen (CEA) has been reported in benign and malignant epithelium of the gall bladder, although cross-reactivity with CEA-related antigens cannot be excluded. We have investigated the immunoreactivity of three monoclonal and two polyclonal antisera to CEA in benign and malignant epithelium of the gall bladder, extrahepatic bile ducts, and ampulla of Vater. The results varied with the antisera, due to cross-reactivity with CEA-related antigens and according to the epitope specificities. Little CEA was found in benign epithelium using monoclonal antisera or following absorption of a polyclonal antiserum from Dako with human liver powder. The latter was the most reliable at detecting malignant epithelium of the gall bladder and extrahepatic bile ducts. All antisera showed equal immunoreactivity in tumours of the ampulla of Vater. The differences seen in the immunoreactivities between tumours of the upper and lower extrahepatic biliary tract probably indicate a variation in the occurrence of CEA epitopes.

Carcinoma of the ampulla of Vater: expression of cancer-associated antigens inversely correlated with prognosis.

To obtain some useful pathologic indicators for predicting the prognosis in carcinomas of the ampulla of Vater, we analyzed 24 surgically resected ampullary carcinomas pathologically with immunohistochemistry of cancer-associated antigens. Pancreatic invasion, lymph node metastasis, and histology of the tumor were significantly correlated with poor prognosis (p less than 0.01), but the size or ulceration of the tumor did not significantly affect the prognosis (p less than 0.05). Immunohistochemically, diffuse positivity for anti-CA19-9 monoclonal antibody was demonstrated in 10 carcinomas and that for anti-carcinoembryonic antigen (CEA), in 10. Eight of them showed synchronously diffuse immunoreactivities for both antigens. Although there was no significant correlation between diffuse positivity for CA19-9 and pathologic factors, CA19-9-positive cases exhibited significantly poor prognoses (p less than 0.01). Diffuse positivity for CEA was correlated with pancreatic invasion (p less than 0.05) and poor prognosis (p less than 0.05). Immunohistochemical study of cancer-associated antigens may disclose some malignant potential of ampullary carcinoma other than that expressed in the morphology. Furthermore, because of the consistency of staining results, immunohistochemistry of cancer-associated antigens may also be useful in predicting preoperatively the prognosis of ampullary carcinoma in biopsied materials.