Julie Earl

Anticipation in familial pancreatic cancer

Background: Previous studies of anticipation in familial pancreatic cancer have been small and subject to ascertainment bias. Our aim was to determine evidence for anticipation in a large number of European families. Patients and methods: A total of 1223 individuals at risk from 106 families (264 affected individuals) were investigated. Generation G3 was defined as the latest generation that included any individual aged over 39 years; preceding generations were then defined as G2 and G1. Results: With 80 affected child-parent pairs, the children died a median (interquartile range) of 10 (7, 14) years earlier. The median (interquartile range) age of death from pancreatic cancer was 70 (59, 77), 64 (57, 69), and 49 (44, 56) years for G1, G2, and G3, respectively. These indications of anticipation could be the result of bias. Truncation of Kaplan-Meier analysis to a 60 year period to correct for follow up time bias and a matched test statistic indicated significant anticipation (p = 0.002 and p<0.001). To minimise bias further, an iterative analysis to predict cancer numbers was developed. No single risk category could be applied that accurately predicted cancer cases in every generation. Using three risk categories (low with no pancreatic cancer in earlier generations, high with a single earlier generation, and very high where two preceding generations were affected), incidence was estimated without significant error. Anticipation was independent of smoking. Conclusion: This study provides the first strong evidence for anticipation in familial pancreatic cancer and must be considered in genetic counselling and the commencement of secondary screening for pancreatic cancer.

Benefit of Surveillance for Pancreatic Cancer in High-Risk Individuals: Outcome of Long-Term Prospective Follow-Up Studies From Three European Expert Centers

PURPOSE Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Hereditary factors play a role in the development of PDAC in 3% to 5% of all patients. Surveillance of high-risk groups, may facilitate detection of PDAC at an early stage. The aim of this study was to assess whether surveillance aids detection of early-stage PDAC or precursor lesions (PRLs) and improves the prognosis. PATIENTS AND METHODS Screening outcomes were collected from three European centers that conduct prospective screening in high-risk groups including families with clustering of PDAC (familial pancreatic cancer [FPC]) or families with a gene defect that predisposes to PDAC. The surveillance program consisted of annual magnetic resonance imaging, magnetic resonance cholangiopancreatography, and/or endoscopic ultrasound. RESULTS Four hundred eleven asymptomatic individuals participated in the surveillance programs, including 178 CDKN2A mutation carriers, 214 individuals with FPC, and 19 BRCA1/2 or PALB2 mutation carriers. PDAC was detected in 13 (7.3%) of 178 CDKN2A mutation carriers. The resection rate was 75%, and the 5-year survival rate was 24%. Two CDKN2A mutation carriers (1%) underwent surgical resection for low-risk PRL. Two individuals (0.9%) in the FPC cohort had a pancreatic tumor, including one advanced PDAC and one early grade 2 neuroendocrine tumor. Thirteen individuals with FPC (6.1%) underwent surgical resection for a suspected PRL, but only four (1.9%) had high-risk lesions (ie, high-grade intraductal papillary mucinous neoplasms or grade 3 pancreatic intraepithelial neoplasms). One BRCA2 mutation carrier was found to have PDAC, and another BRCA2 mutation carrier and a PALB2 mutation carrier underwent surgery and were found to have low-risk PRL. No serious complications occurred as consequence of the program. CONCLUSION Surveillance of CDNK2A mutation carriers is relatively successful, detecting most PDACs at a resectable stage. The benefit of surveillance in families with FPC is less evident.

Palladin Mutation Causes Familial Pancreatic Cancer: Absence in European Families

We read with interest the article published in PLoS Medicine by Pogue-Geile et al. [1] reporting an apparent mutation in the KIAA0992 splice variant of the palladin gene in a family previously reported to have a high incidence of pancreatic cancer. Pogue-Geile and others had previously established that the 4q32–34 locus segregated with pancreatic cancer in this family by screening for pre-neoplastic lesions, which could then be used as a marker for mutation carriers [2]. In the PLoS Medicine paper the authors show that the mutation in palladin is on the 4q32–34 haplotype that segregates with the disease. The European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer (EUROPAC) and the German National Case Collection for Familial Pancreatic Cancer (FaPaCa) have recently shown that a mutation on 4q32–34 is unlikely to explain pancreatic cancer in a majority of our European families, but we did not rule out segregation with the disease in a minority of families [3]. Naturally we were keen to establish if the mutation seen in Family X from America was seen in any of our families, and so we have sequenced the locus in 74 individuals who were either affected by pancreatic cancer or who are obligate carriers (assuming autosomal dominant inheritance) of the disease mutation (in 74 families). We have also sequenced the locus in 14 affected individuals from 14 families with familial multiple mole melanoma with cases of pancreatic cancer (FAMMM-PC) [4] and nine sporadic pancreatic cancer patients of less than 50 years of age. We did not identify the mutation in any of the individuals, neither as a heterozygote or a homozygote. This does not of course mean that other mutations in coding or non-coding regions of this variant of palladin or other variants are absent from European families. However, it is noteworthy that the phenotype of Family X is significantly different from the phenotype common to the families on the EUROPAC/FaPaCa registries. In particular, the incidence of diabetes in our families is relatively low, except where the diabetes is a direct consequence of development of cancer [3]. This presentation contrasts strongly with the family harbouring the palladin mutation [1,2], where diabetes was common. It is possible that Family X (and the association with palladin mutation) is not typical of the familial pancreatic cancer syndrome.

Evaluation of the 4q32-34 Locus in European Familial Pancreatic Cancer

Background: Familial pancreatic cancer (FPC) describes a group of families where the inheritance of pancreatic cancer is consistent with an autosomal-dominant mode of inheritance. The 4q32-34 region has been previously identified as a potential locus for FPC in a large American family. Methods: The region was allelotyped in 231 individuals from 77 European families using nine microsatellite markers, and haplotyping was possible in 191 individuals from 41 families. Families were selected based on at least two affected first-degree relatives with no other cancer syndromes. Results: Linkage to most of the locus was excluded based on LOD scores less than −2.0. Eight families were excluded from linkage to 4q32-34 based on haplotypes not segregating with the disease compared with a predicted six to seven families. Two groups of families were identified, which seem to share common alleles within the minimal disease-associated region of 4q32-34, one group with an apparently earlier age of cancer death than the other pancreatic cancer families. Four genes were identified with potential tumor suppressor roles within the locus in regions that could not be excluded based on the LOD score. These were HMGB2, PPID, MORF4, and SPOCK3. DNA sequence analysis of exons of these genes in affected individuals and in pancreatic cancer cell lines did not reveal any mutations. Conclusion: This locus is unlikely to harbor a FPC gene in the majority of our European families. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1948–55)

Analysis of Methanogen Diversity in a Hypereutrophic Lake Using PCR-RFLP Analysis of mcr Sequences

The incidence and diversity of methanogens in Priest Pot, a dynamic and active lake, were monitored by analysing mcrA gene sequences generated from total DNA samples obtained at different times of the year and amplified using the polymerase chain reaction. A number of mcrA clones were analysed by developing an RFLP-based protocol to generate a number of restriction patterns that were assigned to a number of classes. The RFLP patterns for each class were compared with published sequence information for mcrA from cultured methanogens as well as with those from other experimental studies. They could be used to assign tentative identification for some of the Priest Pot clones and also revealed the presence of a number of clones that could not be affiliated to any known methanogens. The limitations of using RFLP profiles of mcrA gene sequences for studying methanogen ecology are discussed.

Refinement of screening for familial pancreatic cancer

Objective Surveillance programmes are recommended for individuals at risk (IAR) of familial pancreatic cancer (FPC) to detect early pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC). However, the age to begin screening and the optimal screening protocol remain to be determined. Methods IAR from non-CDKN2A FPC families underwent annual screening by MRI with endoscopic ultrasonography (EUS) in board-approved prospective screening programmes at three tertiary referral centres. The diagnostic yield according to age and different screening protocols was analysed. Results 253 IAR with a median age of 48 (25–81) years underwent screening with a median of 3 (1–11) screening visits during a median follow-up of 28 (1–152) months. 134 (53%) IAR revealed pancreatic lesions on imaging, mostly cystic (94%), on baseline or follow-up screening. Lesions were significantly more often identified in IAR above the age of 45 years (p<0.0001). In 21 IAR who underwent surgery, no significant lesions (PDAC, pancreatic intraepithelial neoplasia (PanIN) 3 lesions, high-grade intraductal papillary mucinous neoplasia (IPMN)) were detected before the age of 50 years. Potentially relevant lesions (multifocal PanIN2 lesions, low/moderate-grade branch-duct IPMNs) occurred also significantly more often after the age of 50 years (13 vs 2, p<0.0004). The diagnostic yield of potentially relevant lesions was not different between screening protocols using annual MRI with EUS (n=98) or annual MRI with EUS every 3rd year (n=198) and between IAR screened at intervals of 12 months (n=180) or IAR that decided to be screened at ≥24 months intervals (n=30). Conclusions It appears safe to start screening for PDAC in IAR of non-CDKN2a FPC families at the age of 50 years. MRI-based screening supplemented by EUS at baseline and every 3rd year or when changes in MRI occur appears to be efficient.

Biomarkers for early diagnosis of pancreatic cancer

Pancreatic ductal adenocarcinoma is an aggressive malignancy with a 5-year survival rate of approximately 5%. The lack of established strategies for early detection contributes to this poor prognosis. Although several novel candidate biomarkers have been proposed for earlier diagnosis, none have been adopted into routine clinical use. In this review, the authors examine the challenges associated with finding new pancreatic cancer diagnostic biomarkers and explore why translation of biomarker research for patient benefit has thus far failed. The authors also review recent progress and highlight advances in the understanding of the biology of pancreatic cancer that may lead to improvements in biomarker detection and implementation.

New targeted approaches against the ubiquitin-proteasome system in gastrointestinal malignancies.

The ubiquitin–proteasome system plays a role in a broad range of cellular functions, including cell growth and proliferation. The dysregulation of the ubiquitination process may lead to tumor development. Bortezomib was the first proteasome inhibitor demonstrating activity either as a single agent or in combination with cytotoxic drugs in a wide spectrum of hematological and solid malignancies. A deeper knowledge of the intrinsic molecular mechanisms that govern the ubiquitin system will uncover more opportunities for therapeutic intervention. In this sense, there are a number of compounds under clinical development that target the E3-ubiquitin ligase family, the deubiquitinating enzymes or the enzymatic machinery of the proteasome. In this article we review the rationale for the use of novel ubiquitin–proteasome system inhibitors in gastrointestinal malignancies.

Development of temporal temperature gradient electrophoresis for characterising methanogen diversity.

Temporal temperature gradient electrophoretic (TTGE) analysis of 16S rDNA sequences was optimized to monitor the methanogen population present in water and sediments of a small eutrophic lake, Priest Pot, in the English Lake district. The production of nonrepresentative TTGE profiles due to the generation of polymerase chain reaction (PCR) artifacts initially proved problematical. The use of a proofreading polymerase in the PCR was found to be essential and fully optimized protocols were established and tested to ensure confidence that the TTGE profiles truly reflected sequence diversity. TTGE analysis revealed the methanogen population to be less diverse in water than in sediment. The most genetic diversity was observed in TTGE profiles of sediment DNA isolated in winter and the least was in sediment DNA isolated in summer. DNA sequencing analysis of bands recovered from TTGE gels revealed the presence of two methanogen communities. One clustered with Methanosaeta species and the other with the Methanomicrobiales. Many sequences showed low DNA sequence similarity to known methanogens, suggesting that Priest Pot harbors previously undescribed methanogen species.

Single-Nucleotide Polymorphism (SNP) Analysis to Associate Cancer Risk

Identification of hereditary factors that predispose to cancer allows targeted cancer screening and better quantification of environmental risk factors. The ability to identify which single nucleotide polymorphisms (SNPs) are associated with cancer or segregate with disease in families allows high-risk loci to be identified. In this chapter, two platforms for analysing SNPs are discussed, the Affymetrix and Illumina systems. Application of both platforms requires the same principles of good laboratory practice but there are important differences in materials and methods, which will be discussed.