Loss Of DPC4 Research Articles

Immunohistochemical Loss of DPC4 in Tumors With Mucinous Differentiation Arising in or Involving the Gynecologic Tract.

DPC4 immunohistochemistry (IHC) is usually part of the work-up of mucinous neoplasms in the ovary where the distinction between an ovarian primary and metastatic pancreaticobiliary adenocarcinoma (PanACa) must be made. Although DPC4 IHC is lost in about 55% (46%-61%) of PanACas and typically retained in most primary ovarian mucinous neoplasms, no study has evaluated the expression of this marker in a large cohort of neoplasms arising in or involving gynecologic (GYN) organs. In this study, we retrospectively analyzed the expression of DPC4 IHC in a total of 251 tumors and lesions related to the GYN tract in which DPC4 IHC stain was performed during the initial pathology evaluation. Of these, 138 were primary GYN tumors and lesions, 31 were metastatic GYN tumors involving non-GYN sites, and 83 were metastatic non-GYN tumors involving the GYN tract. We identified 27 cases with loss of DPC4 IHC expression of which 20 cases met the inclusion criteria (i.e. clinical information was available to determine the site of tumor origin). We observed that loss of DPC4 nuclear expression was most commonly seen in tumors of endocervical origin (n=7), of which 5 were gastric-type cervical adenocarcinomas (GCxACa) and 2 were usual-type cervical adenocarcinomas, either primary or metastatic. This was followed by tumors of the pancreaticobiliary tract (n=5), ovary (n=2), and appendix (n=1). In addition, 1 gastric-type vaginal adenocarcinoma (GVaACa) also showed loss of DPC4. Our findings indicate that in female patients with mucinous neoplasms involving the ovary or other sites, with loss of DPC4 by IHC, and negative pancreaticobiliary imaging, the possibility of an occult GCx/GVaACa, and rarely an ovarian primary must be considered.

The expression and underlying angiogenesis effect of DPC4 and VEGF on the progression of cervical carcinoma.

The present study aimed to investigate the expression and roles of deleted in pancreatic carcinoma locus 4 (DPC4) and vascular endothelial growth factor (VEGF) in the development of cervical carcinoma. A total of 115 patients aged between 25 and 60 years were involved, including 19 cervical inflammation, 35 cervical intraepithelial neoplasia (CIN), and 61 cervical squamous-cell carcinoma (CSCC). The protein expression rates of DPC4 and VEGF in all samples were detected using immunohistochemistry. The protein levels of DPC4 and VEGF in CSCC samples were measured using ELISA. Microvessel density (MVD) of each CSCC sample was measured according to the Winder method. Association analysis between DPC4, VEGF and thrombospondin-1 (TSP-1) was conducted using Spearman's correlations. The negative expression rate of DPC4 [DPC4 (-)] and positive expression rate of VEGF [VEGF (+)] of the CSCC group were significantly higher compared with that in the cervical inflammation and CIN groups (P<0.05). In the CSCC group, the protein level of DPC4 decreased, while the VEGF level increased significantly compared with the healthy control group (P<0.05). The MVD in the DPC4 (-), VEGF (+) and TSP-1 (-) groups was significantly increased compared with that of the DPC4 (+), VEGF (-), and TSP-1 (+) groups (P<0.05). The expression of DPC4 was negatively associated with VEGF and TSP-1 (P<0.01). These results suggest that DPC4, VEGF and TSP-1 are involved in the carcinogenesis of cervical carcinoma by inducing angiogenesis. In addition, the loss of DPC4 induces angiogenesis through increasing VEGF. Thus, VEGF may be a target gene regulated by DPC4.

Molecular Aberrations in Periampullary Carcinoma.

Periampullary carcinomas are a group of rare lesions around the ampulla of Vater including distal bile duct and duodenum and are very different from pancreatic ductal adenocarcinoma clinically and pathologically, but the molecular alterations in these tumours are less known. Genetic alterations of the KRAS oncogenes, tumour suppressor genes p53, p16 and MADH4 (SMAD4/DPC4) and genome maintenance genes (MLHI, MSH2) are commonly altered in pancreatic adenocarcinoma and have also been described in periampullary cancers, although at lower frequencies. To understand the molecular characteristics of non-pancreatic periampullary carcinomas, ampullary cancers can now be further defined accurately into their intestinal and pancreatobiliary subtypes using histomolecular profiling. KRAS mutation, which occurs in most pancreatic cancers, is found to occur less frequently in ampullary (42-52%), biliary (22-23%) and duodenal cancers (32-35%). Mutations are also found in tumour suppressor genes (p53) and are associated with transformation of adenomas and low-grade carcinomas into high-grade carcinomas. Loss of DPC4 occurs late in ampullary carcinogenesis. This study summarizes the current knowledge in molecular aberrations in non-pancreatic periampullary cancers.

Adenocarcinoma of the Minor Duodenal Papilla and Its Precursor Lesions

The minor duodenal papilla drains the accessory pancreatic duct of Santorini and lies proximal to the ampulla of Vater. Adenocarcinoma and its precursor lesions arising in the minor papilla are rare. Literature data thus far are limited to a few individual case reports, and the condition is consequently poorly defined. Our study cases were composed of carcinomas fulfilling all of the following criteria: location at 1.5 to 2.5 cm proximal to the major papilla; presence of associated submucosal pancreatobiliary-type ducts with periductal glands or acinar tissue; a predominant submucosal location of the tumor; and lack of an intestinal-type adenoma in the adjacent duodenal mucosa. Tumors were studied morphologically, immunohistochemically, and clinically. Nine cases fulfilling the inclusion criteria were identified. There were 5 men and 4 women with an age range of 50 to 76 years (median, 72 y). The tumor size ranged from 1.2 to 4.4 cm (median, 3 cm). The carcinomas were of colloid type (3 tumors), pancreatobiliary type (4), or nonmucinous intestinal type (2). Five cases were associated with an intraductal papillary mucinous neoplasm (IPMN)-like precursor lesion within the residual structures of the minor papilla in the duodenal submucosa. Immunohistochemically, the intestinal-type and mucinous-type tumors tended to be positive for CK20, CDX2, MUC2, and B72.3, and pancreatobiliary-type tumors tended to be positive for CK7, MUC1, B72.3, and CA125. Loss of DPC4 (Smad4) expression was found in the pancreatobiliary-type carcinomas only. Two tumors showed loss of DNA mismatch-repair protein expression, one losing MLH1 and PMS2 and the other losing MSH6. Both patients were older than 60 years, and neither had germline mutation testing. Follow-up information was available for 6 patients (median follow-up time, 67.5 mo): 3 of the 6 patients died of disease at 60, 75, and 85 months after surgery, respectively, and all 3 patients had an intestinal-type carcinoma (1 colloid and 2 nonmucinous). The patient whose tumor was MSH6 deficient was alive without evidence of disease 51 months after surgery. In conclusion, adenocarcinomas of the minor papilla are rare tumors occurring predominantly in the sixth to seventh decade. Some of them arise from IPMN-like precursors in the residual submucosal minor papilla tissue. Morphologically, immunohistochemically, and clinically they are similar to ampullary or IPMN-associated pancreatic carcinomas and can exhibit either an intestinal, colloid, or pancreaticobiliary phenotype. DNA mismatch-repair deficiency may occur. A careful gross and histologic examination is essential to accurately recognize the site of origin of minor papilla carcinomas.

MP28-08 DPC4 LOSS OF EXPRESSION IN INVASIVE UROTHELIAL CARCINOMAS: AN IMMUNOHISTOCHEMICAL STUDY

INTRODUCTION AND OBJECTIVES: DPC4 (SMAD4) is a tumor suppressor gene that encodes for a critical transcription factor involved in the TGF-beta signal pathway. Genetic inactivation of the DPC4 gene has been described in pancreatic and extrahepatic biliary adenocarcinomas, and loss of DPC4 expression by immunohistochemistry is considered a relatively specific marker of tumors originating from those sites. However, the status of DPC4 has not been previously studied in urothelial carcinomas (UC). We evaluated DPC4 expression in a set of invasive UC of the bladder and the upper urinary tract (UT). METHODS: Formalin-fixed paraffin-embedded tissues from 13 invasive UC of the upper UT (12 high grade and 1 low grade) and 18 invasive high grade UC of the bladder were retrieved from our surgical pathology archives and used to build 2 tissue microarrays. Paired tumor and non-neoplastic tissue were spotted 4 times each. DPC4 expression was evaluated using standard immunohistochemistry (DPC4: clone B8, Santa Cruz, CA). DPC4 expression in each case was categorized as intact (positive DPC4) or lost (at least focal negative DPC4). Only spots with positive internal control were considered. Intact labeling was defined as uniform expression of DPC4 in the cytoplasm of tumor cells per spot, with at least focal nuclear expression of DPC4. DPC4 loss was defined as lack of both cytoplasmic and nuclear expression in any subset of tumor cells in the presence of positive internal control staining. Several cut off values were used to reflect the extent of DPC4 loss ( 75% of the tumor cells). RESULTS: We found varying extent of DPC4 loss in 14 cases of invasive UC. Among the bladder carcinomas, 7/18 (39%) cases had some extent of DPC4 loss. Among the upper UT carcinomas, 7/13 (54%) cases had some extent of DPC4 loss, as detailed in table 1. Intact DPC4 staining was observed in 11/18 cases (61%) of invasive UC of the bladder and in 6/13 cases (46%) of invasive UC of upper UT. CONCLUSIONS: Our findings reveal that loss of DPC4 expression can be observed in UC. The current TMA based findings merrit further evaluation in whole sections of UC using immunohistochemistry. If confirmed, our findings suggest that urothelial carcinoma should be considered in the differential diagnosis of a metastatic carcinoma of unknown primary, when loss of DPC4 is seen. Sequencing analysis to assess DPC4 gene status in UC is also warranted.

Abstract 5318: DPC4 loss results in the activation of alternative oncogenic pathways in pancreatic cancer

Abstract Introduction: Pancreatic cancer remains one of the most lethal forms of cancer, with a five-year survival rate of less than five percent. Many of these patients succumb to complications from metastasis. We have shown that genetic inactivation of DPC4, a major factor in TGF-β signaling, is significantly correlated with widespread metastasis in pancreatic cancer patients at autopsy. We hypothesize that DPC4 loss may be driving pancreatic cancer metastasis by forcing the TGF-β signal down an alternative pathway. Methods: DPC4 was stably restored into three cell lines that harbor a homozygous deletion of the locus (A2.1, A6L, and BxPC3), forming isogenic pairs of DPC4−/− and DPC4+ cells. Matched DPC4−/− and DPC4+ cells were treated with TGF-β1 for 24 hours prior to protein extraction. Immunoblotting was performed for total and phospho Akt (Pi3K pathway), p44-42 (ERK pathway), and Stat3 (JAK-Stat pathway), as well as nuclear and cytoplasmic p65 (NFκB pathway), as these networks have previously been identified as being activated by TGF-β. Immunohistochemistry was performed on 50 surgically resected pancreatic cancers for pAkt. Results: DPC4 was successfully introduced into DPC4−/− cells, as evidenced by semi-quantitative PCR for DPC4, as well as a TGF-β luciferase assay. DPC4+ cells displayed characteristic differences in cell behavior, such as decreased proliferation, migration, and invasion, compared to DPC4−/− cells. Western blot analysis of Smad-independent TGF-β pathways in three sets of DPC4-complemented pancreatic cancer cell lines revealed patterns of preferential activation of the Pi3K and ERK signaling pathways in cells lacking DPC4. In surgically-resected pancreatic cancers, activation of Pi3K was associated with the presence of lymph node metastases (p=0.03), while concomitant DPC4 loss and Pi3K pathway activity was associated with poor survival in pancreatic cancer patients (p=0.02). Conclusions: Restoration of DPC4 into DPC4−/− cells results in the abrogation of Pi3K and ERK signaling, and simultaneous DPC4 loss and Pi3K activation correlates to a poor prognosis of pancreatic cancer patients at surgery. Further studies will confirm whether genotype-specific activation of the ERK and Pi3K pathways plays a role in pancreatic cancer cell behavior, particularly those processes that directly impact metastasis (i.e. migration and invasion). Nevertheless, it is intriguing to consider the Pi3K and ERK pathways as therapeutic targets in pancreatic cancer patients with loss of DPC4. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5318. doi:1538-7445.AM2012-5318

Correlation of DPC4 status with outcomes in pancreatic adenocarcinoma patients receiving adjuvant chemoradiation.

168 Background: In an autopsy series of patients with advanced pancreatic cancer (PCA), loss of DPC4 was highly correlated with disseminated metastasis. The purpose of this study was to determine if DPC4 gene status predicts for survival and patterns of recurrence following adjuvant (adj) chemoradiation (CRT). Methods: 101 patients who underwent surgery followed by adjuvant 5-FU or gemcitabine-based CRT were studied. Imaging studies were reviewed to assess patterns of recurrence and tumor tissue obtained to determine DPC4 immunolabeling status. DPC4 status was graded as intact or lost/mutated. Kaplan-Meier estimates were used to compute survival and Cox proportional hazards were used to compare risk factors. Results: Median overall (mOS) and progression-free survival (PFS) was 22.6 mos (95% CI 18.8 to 31.6) and 14.0 mos (95% CI 11.5 to 18.4). The mOS and 1-yr OS for patients with DPC4 intact vs. lost status was 21.9 mos (95% CI 16.8-32.4) and 78.4% vs. 22.6 mos (95% CI 18.4-32.6) and 78.2%, respectively (HR: 1.05, p=0.82). After adjusting for node, margin status, tumor grade, tumor size, and age, DPC4 status did not predict for mOS (RR 1.04, 95% CI: 0.62-1.74, p=0.89). Time to first progression at any site for PCA with DPC4 intact vs. lost status was 13.8 vs. 14.0 mos (p=0.79). Local recurrence was more common in PCA with DPC4 loss than with intact status (34.4% vs. 13.7%, p=0.012). There was no difference in the rates of distant recurrence in PCA with intact vs. loss of DPC4 expression (62.8% vs. 55.7%, p=0.45); however, DPC4 loss was more commonly associated with liver recurrence (27.9% vs. 19.6%, p=0.31). Conclusions: In pancreatic cancer patients receiving adj CRT, loss of DPC4 labeling in their resected PCA indicates a greater likelihood of developing local recurrence despite having received adj CRT. Efforts to improve loco-regional control are therefore needed for these patients following surgery and adj CRT. No significant financial relationships to disclose.

Abstract 1613: BTK as a drug target for cancers that harbor inactivating mutations in the DPC4 gene

Abstract The Deleted in Pancreatic Cancer locus 4 (DPC4) is a tumor suppressor gene that is often inactivated in pancreatic and colorectal cancers. In pancreatic adenocarcinomas, the frequency of inactivation of DPC4 is approximately 55%, while in colorectal carcinomas has been reported to be 10% to 35%. The loss of DPC4 has been shown to be associated with the progression and malignancy of pancreatic cancer along with decreased patient survival. We employed a small interfering (siRNA) library based screening strategy to identify potential synthetic lethal partners of the DPC4 gene. We used a kinase focused siRNA library that consisted of two siRNA oligonucleotides for each of the 624 protein kinase genes. A DPC4 isogenic pair, BxPC3-Vector and BxPC3-DPC4 (Wang et al. 2006) were treated by the siRNA oligonucleotides in parallel and the effects of the siRNA oligonucleotides on the growth of the cell lines were then compared. siRNA oligonucleotides that selectively inhibited the cell growth of the BxPC3-Vector cell line were selected as potential positive hits. These genes, once validated, represented potential drug targets that are very specific to cancer cells harboring mutations in the DPC4 gene. One of the top-ranked hits we identified from this screening is the Bruton agammaglobulinemia tyrosine kinase (BTK). With BTK siRNA treatment, BxPC3-Vector cells, which are DPC4 null, showed ∼30% more cell growth inhibition than the BxPC3-DPC4 cells, which are DPC4 wildtype. This selectivity of BTK siRNA against DPC4 null cells was further confirmed in a confirmation screen using two pancreatic cancer cell lines, BxPC3 and PANC-1, which are DPC4 null and DPC4 wildtype, respectively. BTK siRNA also showed significant selectivity against DPC4 deficiency in another pair of DPC4 isogenic colon cancer cell lines, HCT-116 and HCT-116-DPC4-knockout. To validate the BTK siRNA selectivity findings, we evaluated the BTK inhibitor PCI-32765 in our cancer cell line models and observed the same selectively against DPC4 null pancreatic and colon cancer cell lines. These results indicate that BTK is a potential molecular target for pancreas, colon, and other cancers harboring inactivating mutations in the DPC4 gene. (The work was supported by a NCI grant P01CA109552.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1613.

Primary Pancreatic Carcinoma or Metastasis from Ovary? Solutions for a Diagnostic Dilemma

Purpose: Introduction: Carcinoma of unknown origin (CUO) accounts for 3-5% of all human malignancies and presents as a diagnostic challenge. We describe a case of pancreatic adenocarcinoma with metastasis to the ovary which presented as CUO. We emphasize the importance of immuno-histochemistry (IHC) as a valuable tool to help localize the primary site. Case Report: 44 year old woman presented with nausea, vomiting, decreased appetite, weight loss and obstructive jaundice. She had a tender right upper quadrant mass and a left adnexal mass. Elevation in both CA 19-9 and CA 12-5 levels (to 449 and 198, respectively) along with presence of 1.7 cm mass at the tail of pancreas and 13 cm multiseptate left ovarian mass on imaging raised suspicion for presence of both pancreatic and ovarian malignancy. Subsequently, patient underwent hysterectomy, bilateral salphingo-oophorectomy and core needle biopsy of pancreatic tail. Histology was positive for adenocarcinoma. The IHC showed that both the ovarian tumors cells and the pancreatic tumor cells were positive for CK7 and negative for CK20, CDX2, p16, Dpc4. This confirmed the primary tumor was pancreatic adenocarcinoma with metastasis to ovary. Discussion: Differential diagnosis in the present case are synchronously detected primary pancreatic and ovarian adenocarcinoma, primary ovarian malignancy with metastasis to pancreas or vice versa. Huge size of left ovarian tumor along with elevated CA 12-5 raised suspicion for primary ovarian carcinoma while MRI images (metastatic tumors to pancreas usually tend to be multiple and hypervascular) with elevated CA 19-9 supported a primary pancreatic cancer. Metastatic pancreatic carcinoma to ovaries with their tendency for large growth and cystic change with morphology showing mucinous epithelium with cystadenomatous growth pattern, are notorious in simulating ovarian mucinous cystic neoplasms (MCN) and represents a particular challenge. IHC was critical for diagnosing primary malignancy in this patient. Ovarian MCNs express CDX-2, CK-7 and CK20 while pancreatic MCN are positive for CK7, CEA, CAM5.2 and EMA. Our patient had negative staining for CDX-2 and CK-20 along with positive staining for CK-7 and CEA suggestive of primary pancreatic malignancy. It has been shown that with the loss of DPC4, metastatic pancreatic carcinoma is more likely, which helped confirm the diagnosis in our patient. Conclusion: This case brings forth the diagnostic dilemma in the localization of the primary site in CUO which is especially true when pancreatic cancer metastasizes to ovary. It emphasizes the importance of newer IHC techniques which can be very helpful as shown in our case and henceforth allows for appropriate staging and treatment.

The pathology and genetics of metastatic pancreatic cancer.

Abstract Context.—Metastatic disease is the most critical determinant of resectability of pancreatic cancer and accounts for the poor outcome of patients with this disease. Thus, a better understanding of metastatic pancreatic cancer will afford new opportunities for therapeutic intervention. Objective.—To summarize and discuss the current understanding of the clinical and molecular features of metastatic pancreatic cancer. Data Sources.—Published literature on advanced stage pancreatic cancer, pancreatic cancer metastasis, and autopsy findings in patients with pancreatic cancer. Conclusions.—In the clinical setting, it can be difficult to distinguish a metastatic pancreatic carcinoma from primary neoplasms in the liver, lung, or ovary. However, immunolabeling for DPC4 protein as part of a diagnostic panel is useful for making this distinction. Emerging data from a variety of investigators now indicate that overexpression of EphA2, loss of DPC4 and MKK4, and aberrant activation of the Hedgehog signaling p...

The Pathology and Genetics of Metastatic Pancreatic Cancer

Metastatic disease is the most critical determinant of resectability of pancreatic cancer and accounts for the poor outcome of patients with this disease. Thus, a better understanding of metastatic pancreatic cancer will afford new opportunities for therapeutic intervention. To summarize and discuss the current understanding of the clinical and molecular features of metastatic pancreatic cancer. Published literature on advanced stage pancreatic cancer, pancreatic cancer metastasis, and autopsy findings in patients with pancreatic cancer. In the clinical setting, it can be difficult to distinguish a metastatic pancreatic carcinoma from primary neoplasms in the liver, lung, or ovary. However, immunolabeling for DPC4 protein as part of a diagnostic panel is useful for making this distinction. Emerging data from a variety of investigators now indicate that overexpression of EphA2, loss of DPC4 and MKK4, and aberrant activation of the Hedgehog signaling pathway are associated with metastatic propensity of pancreatic cancers, providing novel therapeutic targets for the most lethal stage of this disease.

Inhibition of pancreatic carcinoma cell growth in vitro by DPC4 gene transfection

To detect the expression of DPC4 in malignant and non-malignant specimens of human pancreas, and observe the inhibition of retroviral pLXSN containing DPC4 on pancreatic carcinoma cells in vitro. The expression of DPC4 was determined in 40 pancreatic adenocarcinoma and 36 non-malignant pancreatic specimens by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohisto-chemistry. Furthermore, we constructed retroviral vectors containing DPC4, which then infected the pancreatic carcinoma cell line BxPC-3. Cell growth in vitro after being infected was observed, and the vascular endothelial growth factor (VEGF) mRNA level in the daughter cells was determined by semi-quantitative PCR assay. The RT-PCR assay showed a positive rate of DPC4 mRNA in 100% (36/36) of normal specimens, compared to 40% (16/40) in adenocarcinoma specimens. The regional and intense positive cases of DPC4 expression in adenocarcinoma detected by immunohistochemistry were 10 and four, whereas it was all positive expression in normal tissues. There was a significant difference of DPC4 expression between them. The stable expression of DPC4 in the pancreatic carcinoma cells BxPC-3 could be resumed by retroviral vector pLXSN transfection, and could inhibit cell growth in vitro. Rather, DPC4 could decrease VEGF mRNA transcription levels. The deletion of DPC4 expression in pancreatic carcinoma suggests that loss of DPC4 may be involved in the development of pancreatic carcinoma. The retroviral vector pLXSN containing DPC4 can inhibit the proliferation of pancreatic carcinoma cells, and down-regulate the level of VEGF.

Clinicopathologic and Immunohistochemical Study of Small Apparently ???De Novo??? Colorectal Adenocarcinomas

Rarely, adenocarcinomas of the colorectum develop as small (< or =1.0 cm) rapidly invasive tumors without an obvious adenomatous or "in situ" component. These tumors have been termed "de novo" carcinomas. Although it is believed by some that these tumors are more aggressive than conventional large adenocarcinomas with an identifiable in situ component, little is known about the biologic characteristics and natural history of these lesions. The aim of this study was to evaluate and compare the pathologic features, biologic characteristics, and natural history of small apparently de novo invasive colorectal adenocarcinomas with conventional large (>1.0 cm) carcinomas. Routinely processed specimens from 20 patients (M/F ratio: 13/7; mean age: 65 y) with small apparently de novo invasive colorectal adenocarcinomas (all < or =1.0 cm in size) were evaluated for a variety of clinical and pathologic features. In addition, immunostains for p53, beta-catenin, DPC4, hMLH1, hMSH2, and MGMT were evaluated in all cases. The findings in this group of cases were compared with those from 20 control patients (M/F ratio: 8/12; mean age: 60 y) with stage-matched conventional "large" colorectal adenocarcinomas (all >1.0 cm in size). Patients were followed for a mean of 52.6 and 60.6 months, respectively, for the 2 groups. Small apparently de novo invasive adenocarcinomas were present in the left colon, transverse colon, and right colon in 85%, 10%, and 5% of cases, respectively. Their mean size was 7 mm (range: 3 to 10 mm). All cases were stage T1 and the majority were moderately differentiated (75%). Only 1 (5%) patient had lymph node metastases. Two (10%) cases were mucinous and only 1 (5%) showed prominent tumor infiltrating lymphocytes. Upon complete sectioning of the tissue blocks of tumor, residual foci of adenomatous epithelium were present in 16/20 (80%) cases, of which 75% contained foci of high-grade dysplasia. P53 and nuclear beta-catenin staining was present in 70% and 85% of cases, respectively, but only 5 cases (25%) showed loss of DPC4. Loss of MGMT expression was seen in 5 cases (25%), loss of hMSH2 in only 1 case (5%), and none showed loss of hMLH1. Only 2 patients (10%) developed visceral metastases upon follow-up. Control patients had similar demographic features, clinical outcome, anatomic distribution of tumors, degree of differentiation, and prevalence of positivity for the immunostains noted above, to the study cases. In our patient population, true small de novo colorectal adenocarcinomas, tumors that lack an identifiable adenomatous component, are exceedingly rare, because complete tissue sectioning reveals residual adenomatous tissue in the majority of cases. The biologic characteristics and natural history of small carcinomas with a minimal dysplastic component, and those with no identifiable adenomatous component, are similar to conventional large (>1 cm) adenocarcinomas, and, thus, they should probably be treated similarly.

Vascular Endothelial Growth Factor and DPC4 Predict Adjuvant Therapy Outcomes in Resected Pancreatic Cancer

Angiogenesis is important for pancreatic cancer progression, but its role in predicting response to therapy is not known. We investigated the association of various angiogenic factors and intratumoral microvessel density (IMD) with adjuvant therapy and survival in resected pancreatic cancer. Tissue cores from a multi-institutional retrospective series of resected patients were used to build a pancreatic cancer tissue microarray. Vascular endothelial growth factor (VEGF), platelet-derived endothelial cell growth factor (PD-ECGF), CD31 (for IMD), and DPC4 expression were determined using immunohistochemistry. Expression of VEGF and PD-ECGF, both proangiogenic factors, was observed in 70 (56%) and 75 (59%) of 124 tumors, respectively. Expression of DPC4, an angiogenesis inhibitor, was observed in 59 of 124 (48%) tumors. VEGF expression correlated significantly with increased IMD (P=.03), as did loss of antiangiogenic DPC4 (P=.05). PD-ECGF expression did not correlate with IMD. Use of adjuvant therapy was associated with increased survival in patients with VEGF-positive tumors (18.8 [treated] versus 11.2 [untreated] months; hazard ratio [HR]=0.38, 95% confidence interval [CI], 0.19-0.76; P=.005), but not in patients with VEGF-negative tumors. Similarly, improved survival was observed in patients with high IMD (16.3 [treated] versus 11.2 [untreated] months; HR=0.44, 95% CI, 0.23-0.87; P=.02) and in patients with loss of DPC4 (20.3 [treated] versus 11.2 [untreated] months; HR=0.31, 95% CI, 0.14-0.67; P=.002), but not in those with low IMD or normal DPC4 expression. VEGF (stimulator) and DPC4 (inhibitor) are important regulators of pancreatic tumor angiogenesis and predictive of benefit from adjuvant therapy. Adjuvant therapy may have both antiangiogenic and cytotoxic effects. Addition of anti-VEGF agents to adjuvant regimens may further improve outcomes.

Immunohistochemistry Can Help Distinguish Metastatic Pancreatic Adenocarcinomas From Bile Duct Adenomas and Hamartomas of the Liver

Not uncommonly, bile duct adenomas (BDAs) and hamartomas (BDHs) of the liver may be difficult to distinguish from metastatic well-differentiated ductal adenocarcinoma of the pancreas. However, this distinction is critical for proper staging and patient management. The primary purpose of this study was to determine if a panel of immunohistochemical stains can help distinguish BDA or BDH from metastatic pancreatic adenocarcinoma in the liver. Routinely processed tissue sections from 25 BDA, 10 BDH, 25 metastatic pancreatic adenocarcinomas to the liver and 6 cases each of metastatic colorectal, breast, and lung adenocarcinomas were immunohistochemically stained for CK7, CK8/CK18, CK19, CK20, p53, p63, TAG-72, monoclonal CEA (mCEA), polyclonal CEA (pCEA), HER-2/neu, AMACR (alpha-methylacyl-CoA racemase), Dpc4 (Smad4), and mesothelin. The slides were evaluated in a blinded fashion, and the results were compared between the benign and malignant lesions. Significantly more (P < 0.05) metastatic pancreatic adenocarcinomas were positive for CK20 (76%), p53 (60%), TAG-72 (88%), mCEA (92%), HER2/neu (40%), and mesothelin (64%) and showed loss of Dpc4 (44%), in comparison to BDA (CK20, 40%; p53, 0%; TAG-72, 0%; mCEA, 0%; HER2/neu, 12%; mesothelin, 0%; loss of Dpc4, 0%) or BDH (CK20, 10%; p53, 0%; TAG-72, 0%; mCEA, 10%; HER2/neu, 0%; mesothelin, 0%; loss of Dpc4, 0%). Of these antibodies, p53, TAG-72, mCEA, loss of Dpc4, and mesothelin had the highest specificity for pancreatic adenocarcinoma, with mCEA having the highest sensitivity (92%). No significant differences were observed in the degree of CK7, CK8/CK18, CK19, or pCEA expression between the three types of lesions. Although none of the BDA or BDH was positive for either p63 or AMACR, two of the metastatic pancreatic adenocarcinomas (8%) were positive for each of these peptides (P > 0.05). For nonpancreatic adenocarcinomas, mCEA showed a reasonably high sensitivity and 100% specificity in the differential diagnosis versus BDA. Immunohistochemical expression of p53, TAG-72, mCEA, mesothelin, and loss of Dpc4 can help distinguish metastatic pancreatic adenocarcinoma in the liver from BDA or BDH. Although p63 and AMACR are also specific for pancreatic adenocarcinoma, their low sensitivity limits their use in clinical practice.

Genetic Alterations in Goblet Cell Carcinoids of the Vermiform Appendix and Comparison with Gastrointestinal Carcinoid Tumors

Goblet cell carcinoid is a relatively rare neuroendocrine tumor of the vermiform appendix with poorly understood molecular pathogenesis. We studied the clinicopathologic features and genetic alterations, including allelic loss of chromosomes 11q, 16q, and 18q; sequencing of the K-ras, β-catenin, and DPC4 (SMAD4) genes; and p53 overexpression and loss of DPC4 by immunohistochemistry; in 16 goblet cell carcinoids. We compared the allelic loss in goblet cell carcinoids to those in 18 gastrointestinal carcinoid tumors. For goblet cell carcinoids, appendiceal perforation was the most common (70%, 7/10) clinical presentation. The mean tumor size was 2.0 ± 1.5 cm (range, 0.4 to 4.5 cm). The tumor invaded to appendiceal serosa in 50% (8/16) of patients, and two patients had metastasis in lymph nodes or adjoining viscera. With mean follow-up of 24 ± 14 months (median, 23 mo), 1 of 10 patients had died of disease, and 2 others had tumor recurrence. All four patients with metastases, recurrences, and/or death from disease had serosal involvement at presentation (P = .02). Loss of heterozygosity of chromosome 11q was present in 25% of goblet cell carcinoids, 14% of ileal carcinoid tumors, and 9% of nonileal carcinoid tumors; of chromosome 16q in 38%, 29%, and 0 (P = .02); and of chromosome 18q in 56%, 86%, and 9% (P = .002), respectively. No mutations of K-ras, β-catenin, or DPC4 genes; p53 overexpression; or loss of staining for DPC4 was present in any tumors. These findings suggest that allelic loss of chromosomes 11q, 16q, and 18q in goblet cell carcinoids and ileal carcinoids may have an important role in the pathogenesis of these tumors.

Immunohistochemical and Genetic Analysis of Non–Small Cell and Small Cell Gallbladder Carcinoma and Their Precursor Lesions

Gallbladder carcinomas can be highly lethal neoplasms. Relatively little is known about the genetic abnormalities that underlie these tumors, particularly with respect to their timing in neoplastic progression. The authors evaluated 5 noninvasive dysplasias and 33 invasive gallbladder carcinomas (6 small cell carcinomas, 27 non–small cell carcinomas, of which 16 were accompanied by an in situ carcinoma component) for expression of the protein products of the p16, p53, Dpc4, and pRB tumor suppressor genes by immunohistochemistry. Neoplasms were also evaluated for the presence of activating K-ras oncogene mutations. Seventy-five percent of non–small cell gallbladder carcinomas demonstrated loss of p16 expression, whereas 63% accumulated high levels of p53. Loss of Dpc4 and pRB expression was less frequent, seen in 19% and 4% of the neoplasms, respectively. Thirty percent of neoplasms harbored activating K-ras mutations. In contrast, 100% of the small cell carcinomas of the gallbladder demonstrated inactivation of the pRB/p16 pathway; 67% showed loss of pRB expression, and the other 33% lost p16 expression. Eighty-three percent of small cell carcinomas accumulated high levels of p53, whereas loss of Dpc4 expression and activating K-ras mutations were not found. Among 15 evaluable in situ components, 13 harbored the same alterations found in the invasive component. Inactivation of p16 and p53 occur in the majority of non–small cell gallbladder carcinomas. Dpc4 inactivation and K-ras mutations occur in a significant minority of cases. pRB loss is uncommon in non–small cell gallbladder carcinoma, but virtually all small cell carcinomas inactivate the p16/pRB pathway, usually by retinoblastoma protein loss. It is noteworthy that all of these alterations occur at the level of carcinoma in situ.

Role of the DPC4 Tumor Suppressor Gene in Adenocarcinoma of the Ampulla of Vater: Analysis of 140 Cases

The K-ras oncogene is activated in approximately 90% of pancreatic adenocarcinomas, and the DPC4 (MADH4/SMAD4) tumor suppressor gene is inactivated in approximately 55% of pancreatic adenocarcinomas. The contributions of these genetic alterations to the development of adenocarcinoma of the ampulla of Vater have not been fully established. One hundred forty surgically resected ampullary adenocarcinomas (76 with associated adenomas with high-grade dysplasia) were immunohistochemically labeled for the DPC4 gene product, and in 85 cases the results were correlated with the status of the K-ras oncogene from previously reported data. The results were correlated with clinical outcome and with other pathologic predictors of prognosis. Complete loss of Dpc4 labeling was identified in 34% (95% confidence interval [CI]: 26%, 43%) of the invasive carcinomas and in none (upper 95% CI: 6%) of the associated adenomas. Focal loss of Dpc4 was seen in three (4%; 95% CI: 1%, 14%) of the areas of high-grade dysplasia. Complete loss of Dpc4 expression was seen in 28/77 intestinal-type tumors, in 17/46 pancreaticobiliary-type tumors, and in 0/10 colloid carcinomas. Activating point mutations in the K-ras gene were identified in 40% of the invasive cancers. There was no correlation between K-ras gene mutations and Dpc4 expression and no correlation between these variables and survival. The overall 5-year survival rate was 38%. Lymph node metastases were associated with shorter survival (P = .03). Loss of Dpc4 expression occurs in approximately one third of invasive ampullary cancers but is not seen in adenomas; thus, loss of Dpc4 expression occurs late in ampullary carcinogenesis. Although ampullary and pancreatic adenocarcinomas share histologic and molecular features, ampullary carcinomas are less likely to show loss of Dpc4 expression or K-ras gene mutations.

Direct Correlation between Proliferative Activity and Dysplasia in Pancreatic Intraepithelial Neoplasia (PanIN): Additional Evidence for a Recently Proposed Model of Progression

A growing body of morphological, clinical, and genetic observations suggests a progression model for pancreatic ductal adenocarcinoma. In this model, pancreatic ducts progress through a series of architectural and cytological changes that define degrees of pancreatic intraepithelial neoplasia (PanIN). Expressed in dividing cells, Ki-67 has been extensively used as a proliferation marker. Its expression in different grades of PanIN has not been well studied. A total of 76 PanINs from 41 patients were histologically graded according to recently established criteria. These PanINs were then immunolabeled with a monoclonal antibody against Ki-67 (Mib-1). Normal ducts and invasive ductal adenocarcinomas were also labeled with the antibody. In 15 normal ducts, only 0.41% of the epithelial cells expressed Ki-67. Ki-67–labeling indices in the increasing grades of PanIN were as follows: PanIN-1A, 0.69%; PanIN-1B, 2.33%; PanIN-2, 14.08%; and PanIN-3, 22.01%. Fifteen invasive ductal adenonocarcinomas showed an average labeling index of 36.99%. The difference in Ki-67 labeling among these groups was statistically significant (P < .0005, Kruskal-Wallis test). This pattern of proliferation provides additional evidence supporting the recently proposed pancreatic progression model. It also correlates well with known molecular changes, such as activating point mutations in the K-ras oncogene and the loss of DPC4 and p16 gene expression. Ki-67 staining may be useful as an adjunct in the diagnosis of precancerous lesions in the pancreas and may provide a reliable way to identify lesions at high risk for the subsequent development of infiltrating carcinoma.

Differing rates of loss ofDPC4 expression and ofp53 overexpression among carcinomas of the proximal and distal bile ducts

Biliary tract carcinomas are clinically heterogeneous. It is not known if molecular heterogeneity underlies the clinical differences. The authors evaluated 128 bile duct carcinomas, 88 of the distal common bile duct and 40 of more proximal origin (28 perihilar carcinomas, 12 intrahepatic carcinomas), immunohistochemically for abnormalities in the expression of the products of the DPC4 and p53 tumor-suppressor genes. Prognostic factors were evaluated in the series of distal bile duct carcinomas for which follow-up information was available. The authors found that a significantly higher percentage of distal bile duct carcinomas (55%) demonstrated loss of DPC4 expression than did the proximal bile duct carcinomas (15%; P < 0.001). They also found that a significantly higher percentage of the distal tumors abnormally expressed the p53 gene product (51% vs. 26%; P < 0.001). Among the distal common bile duct carcinomas, the presence of poorly differentiated histology correlated with decreased survival in multivariate analysis, while labeling for p53 or Dpc4, margin status, lymph node status, and tumor dimension did not correlate significantly with survival. These results demonstrate that abnormalities in DPC4 and p53 gene expression are frequent in distal common bile duct carcinomas, just as they are in pancreatic ductal adenocarcinoma, suggesting that these two tumor types might share a similar molecular pathogenesis. They also show that proximal and distal bile duct carcinomas have different patterns of inactivation of tumor-suppressor genes, indicating that they often arise through different molecular mechanisms likely reflecting their differing etiologies.