Benoit Terris

miR-221 overexpression contributes to liver tumorigenesis

MicroRNA (miRNAs) are negative regulators of gene expression and can function as tumor suppressors or oncogenes. Expression patterns of miRNAs and their role in the pathogenesis of hepatocellular carcinoma (HCC) are still poorly understood. We profiled miRNA expression in tissue samples (104 HCC, 90 adjacent cirrhotic livers, 21 normal livers) as well as in 35 HCC cell lines. A set of 12 miRNAs (including miR-21, miR-221/222, miR-34a, miR-519a, miR-93, miR-96, and let-7c) was linked to disease progression from normal liver through cirrhosis to full-blown HCC. miR-221/222, the most up-regulated miRNAs in tumor samples, are shown to target the CDK inhibitor p27 and to enhance cell growth in vitro. Conversely, these activities can be efficiently inhibited by an antagomiR specific for miR-221. In addition, we show, using a mouse model of liver cancer, that miR-221 overexpression stimulates growth of tumorigenic murine hepatic progenitor cells. Finally, we identified DNA damage-inducible transcript 4 (DDIT4), a modulator of mTOR pathway, as a bona fide target of miR-221. Taken together, these data reveal an important contribution for miR-221 in hepatocarcinogenesis and suggest a role for DDIT4 dysregulation in this process. Thus, the use of synthetic inhibitors of miR-221 may prove to be a promising approach to liver cancer treatment.

Intraductal papillary mucinous tumors of the pancreas confined to secondary ducts show less aggressive pathologic features as compared with those involving the main pancreatic duct

Intraductal papillary mucinous tumors (IPMTs) of the pancreas are rare tumors characterized by a malignant potential. Because of the progress of imaging procedures, smaller cystic pancreatic lesions are now detected and some of them correspond to IPMTs that involve ectatic pancreatic branch ducts but spare the main pancreatic duct. To investigate differences in morphology and clinical behavior of branch and main duct types of IPMT, a surgical series of 43 cases was studied. All pathologic specimens of IPMT, surgically resected in our institution between October 1987 and July 1998, were analyzed. In all cases, the entire pancreatic specimen was systematically examined. IPMT of the branch type was found in 13 (30%) patients, whereas IPMT of main pancreatic duct type that involved the main pancreatic duct and branch ducts was observed in 30 (70%) patients. Patients with IPMT of the branch type were younger (median age, 55 yrs vs 64 yrs), and all but one of the lesions were located in the head and neck of the pancreas (vs 17 of 30 patients with the main duct type). The size of the cysts ranged from 4 to 55 mm, and the major duct showed a mild dilation in most cases. In contrast to the main pancreatic duct type, which showed invasive carcinoma and in situ carcinoma in 11 (37%) of 30 patients and 6 (20%) of 30 patients, respectively, IPMT of the branch type showed significantly less aggressive histologic lesions with five (39%) patients with simple hyperplasia, six (46%) patients with atypical hyperplasia, and two (15%) patients with in situ carcinoma. No invasive carcinoma was observed in this group. IPMT of the branch type occurs in younger patients and is associated with less aggressive histologic features than is the main pancreatic duct type. Our findings raise the difficult issue of clinical management of IPMT of the branch type as a distinctive group.

Focal nodular hyperplasia of the liver: a comprehensive pathologic study of 305 lesions and recognition of new histologic forms.

Atypical histologic variants of focal nodular hyperplasia have been reported and are sometimes difficult to recognize. To characterize the morphologic spectrum of focal nodular hyperplasia, we studied 305 lesions surgically resected from 168 patients. Clinicomorphologic correlations were established by statistical analyses. The patients included 150 women and 18 men (sex ratio, 8:1; median age, 38 years). One hundred twenty-eight (76.2%) patients had solitary lesions, and 40 (23.8%) had 2 to 30 lesions. All 305 lesions measured 1 mm to 19 cm in diameter. Only 49% of these lesions had one to three macroscopic scars. Histologically, 245 (80.3%) lesions were of classical form, and 60 (19.7%) lesions were nonclassical. The latter were classified as focal nodular hyperplasia of telangiectatic form (47 lesions), of mixed hyperplastic and adenomatous form (five lesions), and with atypia of large cell type (eight lesions). Several benign or malignant tumors were found in association with these lesions. This large retrospective series of focal nodular hyperplasia shows the relative incidence of its classical and nonclassical forms. The absence of a central scar could explain the difficult preoperative diagnosis of some of the cases. The morphologic diagnostic criteria in this study require further prospective evaluation.

Value of endoscopic ultrasound guided fine needle aspiration biopsy in the diagnosis of solid pancreatic masses

AIM To assess the feasibility and diagnostic accuracy of endoscopic ultrasound guided fine needle biopsy (EUS-FNAB) in patients with solid pancreatic masses. METHODS Ninety nine consecutive patients with pancreatic masses were studied. Histological findings obtained by EUS-FNAB were compared with the final diagnosis assessed by surgery, biopsy of other tumour site or at postmortem examination, or by using a combination of clinical course, imaging features, and tumour markers. RESULTS EUS-FNAB was feasible in 90 patients (adenocarcinomas, n = 59; neuroendocrine tumours, n = 15; various neoplasms, n = 6; pancreatitis, n = 10), and analysable material was obtained in 73. Tumour size (⩾ or < 25 mm in diameter) did not influence the ability to obtain informative biopsy samples. Diagnostic accuracy was 74.4% (adenocarcinomas, 81.4%; neuroendocrine tumours, 46.7%; other lesions, 75%; p<0.02). Overall, the diagnostic yield in all 99 patients was 68%. Successful biopsies were performed in six patients with portal hypertension. Minor complications (moderate bleeding or pain) occurred in 5% of cases. CONCLUSIONS EUS-FNAB is a useful and safe method for the investigation of pancreatic masses, with a high feasibility rate even when lesions are small. Overall diagnostic accuracy of EUS-FNAB seems to depend on the tumour type.

Prognosis of malignant intraductal papillary mucinous tumours of the pancreas after surgical resection. Comparison with pancreatic ductal adenocarcinoma

Background: Although the prognosis in malignant resectable intraductal papillary mucinous tumours of the pancreas (IPMT) is often considered more favourable than for ordinary pancreatic ductal adenocarcinoma, the long term outcome remains ill defined. Aims: To assess prognostic factors in patients with malignant IPMT after surgical resection, and to compare long term survival rates with those of patients surgically treated for ductal adenocarcinoma. Methods: Seventy three patients underwent surgery for malignant IPMT in four French centres. Clinical, biochemical, and pathological features and follow up after resection were recorded. Patients with invasive malignant IPMT were matched with patients with pancreatic ductal adenocarcinoma, according to age and TNM stages; survival rates after resection were compared. Results: Surgical treatment for IPMT were pancreaticoduodenectomy (n=46), distal (n=14), total (n=11), or segmentary (n=2) pancreatectomy. The operative mortality rate was 4%. IPMT corresponded to in situ (n=22) or invasive carcinoma (n=51). In the latter group, 17 had lymph node metastases. Overall median survival was 47 months. Five year survival rates in patients with in situ and invasive carcinoma were 88% and 36%, respectively. On univariate analysis, abdominal pain, preoperative high serum carbohydrate antigen 19.9 concentrations, caudal localisation, invasive carcinoma, lymph node metastases, peripancreatic extension, and malignant relapse were associated with a fatal outcome. Using multivariate analysis, lymph node metastases were the only prognostic factor (OR 7.5; 95% CI: 3.4 to 16.4). Overall five year survival rate was higher in patients with malignant invasive IPMT compared with those with pancreatic ductal carcinoma (36 v 21%, p=0.03), but was similar in the subset of stage II/III tumours. Conclusions: The prognosis of patients with resected in situ/invasive stage I malignant IPMT is excellent. In contrast, prognosis of locally advanced forms is as poor as in patients with pancreatic ductal adenocarcinoma.

Prognostic factors in patients with endocrine tumours of the duodenopancreatic area

Background—The development of endocrine tumours of the duodenopancreatic area (ETDP) is thought to be slow, but their natural history is not well known. The aim of this study was to determine the factors that influence survival of patients with ETDP. Patients/Methods—Eighty two patients with ETDP (44 non-functioning tumours, 23 gastrinomas, seven calcitonin-secreting tumours, four glucagonomas, three insulinomas, one somatostatinoma) followed from October 1991 to June 1997 were included in the study. The following factors were investigated: primary tumour size, hormonal clinical syndrome, liver metastases, lymph node metastases, extranodular/extrahepatic metastases, progression of liver metastases, local invasion, complete resection of the primary tumour, and degree of tumoral differentiation. The prognostic significance of these factors was investigated by uni- and multi-variate analysis. Results—Twenty eight patients (34%) died within a median of 17 months (range 1–110) from diagnosis. Liver metastases (p = 0.001), lymph node metastases (p = 0.001), progression of liver metastases (p<0.00001), lack of complete resection of the primary tumour (p = 0.001), extranodular/extrahepatic metastases (p = 0.001), local invasion (p = 0.001), primary tumour size ⩾3 cm (p = 0.001), non-functioning tumours (p = 0.02), and poor tumoral differentiation (p = 0.006) were associated with an unfavourable outcome by univariate analysis. Multivariate analysis identified only liver metastases (risk ratio (RR) = 8.3; p<0.0001), poor tumoral cell differentiation (RR = 8.1; p = 0.0001), and lack of complete resection of the primary tumour (RR = 4.8; p = 0.0007) as independent risk factors. Five year survival rates were 40 and 100% in patients with and without liver metastases, 85 and 42% in patients with and without complete resection of primary tumour, and 17 and 71% in patients with poor and good tumour cell differentiation respectively. Conclusion—Liver metastases are a major prognostic factor in patients with ETDP. Progression of liver metastases is also an important factor which must be taken into account when deciding on the therapeutic approach. The only other independent prognostic factors are tumoral cell differentiation and complete resection of the primary tumour.

Expression profiling of microdissected pancreatic adenocarcinomas

Pancreatic ductal adenocarcinoma is characterized by a paucity of neoplastic cells embedded in a densely desmoplastic stroma. Therefore, laser capture microdissection was performed to obtain homogeneous populations of normal and neoplastic ductal cells. These were subjected to a comparative study of gene expression utilizing human cDNA arrays. A variety of dysregulated genes were identified, comprising cell cycle and growth regulators, invasion regulators, signalling and developmental molecules. In addition to genes already found to be overexpressed in pancreatic cancer, such as TIMP1, MMP7, CD59, rhoC and NDKA, we present evidence to implicate genes which have not previously been reported in this tumour type. These include the overexpressed genes ABL2, Notch4 and SOD1, as well as XRCC1, a DNA repair gene whose transcript was found downregulated. Quantitative real-time RT–PCR (QRT–PCR) was employed to confirm differential expression of ABL2, Notch4 and SOD1 and immunohistochemical analysis was used to verify decreased protein expression of XRCC1 using a custom-built pancreatic tissue array. Combining microarray-derived gene expression profiles of pure pancreatic cell populations, QRT–PCR and pancreas-specific tissue arrays therefore proved to be highly informative in elucidating the molecular pathology of this highly malignant tumour type.

Germline-Activating Mutation in the Kinase Domain of KIT Gene in Familial Gastrointestinal Stromal Tumors

The proto-oncogene KIT encodes the receptor tyrosine kinase KIT. Gain-of-function mutations in the juxtamembrane domain of KIT have been reported in human gastrointestinal stromal tumors. In a family with multiple gastrointestinal stromal tumors and diffuse hyperplasia of myenteric plexus layer, we have identified another mutation of KIT, a single base mutation, resulting in the substitution of Glu for Lys(642) in the kinase I domain, and studied its biological effect in a cellular system. The mouse homologue of the human KIT mutant was generated by site-directed mutagenesis and stably transfected into the interleukin-3-dependent Ba/F3 murine cell line. The oncogenic potential of the mutated KIT was assessed in vitro by a proliferation assay and in vivo by transplantation into nude mice. Transfected Ba/F3 cells grew autonomously in absence of growth factors and formed tumors in nude mice. Substitution of Glu for Lys(642) is an oncogenic mutation in the tyrosine kinase domain of KIT. As germline heterozygous mutation, it causes a diffuse hyperplasia of myenteric interstitial cells of Cajal during embryonic development and occurrence of multiple gastrointestinal stromal tumors at adulthood.

Characterization of Gene Expression Profiles in Intraductal Papillary-Mucinous Tumors of the Pancreas

The molecular pathology of precursor lesions leading to invasive pancreatic ductal adenocarcinomas remains relatively unknown. We have applied cDNA microarray analysis to characterize gene expression profiles in a series of intraductal papillary-mucinous tumors (IPMTs) of the pancreas, which represents one of the alternative routes of intraepithelial progression to full malignancy in the pancreatic duct system. Using a cDNA microarray containing 4992 human genes, we screened a total of 13 IPMTs including nine noninvasive and four invasive cases. Expression change in more than half of the tumors was observed for 120 genes, ie, 62 up-regulated and 58 down-regulated genes. Some of the up-regulated genes in this study have been previously described in classical pancreatic carcinomas such as lipocalin 2, galectin 3, claudin 4, and cathepsin E. The most highly up-regulated genes in IPMTs corresponded to three members of the trefoil factor family (TFF1, TFF2, and TFF3). Immunohistochemistry performed on five genes found to be differentially expressed at the RNA level (TFF1, TFF2, TFF3, lipocalin 2, and galectin 3) showed a good concordance between transcript level and protein abundance, except for TFF2. Hierarchical clustering organized the cases according to the dysplastic and invasive phenotype of theIPMTs. This analysis has permitted us to implicate several genes (caveolin 1, glypican 1, growth arrest-specific 6 protein, cysteine-rich angiogenic inducer 61) in tumor progression. The observation that several genes are differentially expressed both in IPMTs and pancreatic carcinomas suggests that they may be involved at an early stage of pancreatic carcinogenesis.

Gene expression profiles of pancreatic cancer and stromal desmoplasia

Gene expression studies were undertaken in normal pancreas and pancreatic adenocarcinomas to determine new candidate genes that can potentially be used as markers of the disease. The characteristic desmoplastic stromal reaction of pancreatic adenocarcinoma greatly hampers expression studies in this tumour type, and usually necessitates time-consuming tissue microdissection for enrichment of the tumour cell population. We show that fine needle aspiration of cancer provides a fast and efficient way of obtaining samples highly enriched in tumour cells with sufficient yields of RNA. Using Atlas cancer cDNA arrays with 588 cancer-related genes, we describe gene expression profiles of normal pancreas, bulk pancreatic tumour tissues and pancreatic tumour aspirates containing more than 95% tumour cells. Analysis of bulk tissue specimens revealed differentially expressed genes belonging predominantly to the stromal component of the tumour. This contrasted with the results obtained from tumour-cell enriched samples. Several genes already described in pancreatic cancer (caspase 8, TIMP1, CD9, IL-13) were also differentially expressed in our study. Furthermore, we found dysregulated expression of genes not previously associated with pancreatic adenocarcinoma, such as Rac 1, GLG1, NEDD5, RPL-13a, RPS9 and members of the Wnt5A gene family. In summary, we present a panel of genes newly identified in the pathogenesis of pancreatic adenocarcinoma and demonstrate that fine needle aspirates of the tumour mass are a convenient source of material for gene expression studies in tumours accompanied by desmoplastic reactions.