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Section of Pathological Anatomy, Department of Pathology and Laboratory Medicine, University of Parma, via Gramsci, 14, I-43100 Parma, Italy1 Department of Medical and Surgical Sciences, University of Padua, Padua, Italy2 Section of Pathology, Department of Pathologic Anatomy and Legal Medicine, University of Modena and Reggio Emilia, Modena, Italy3 Digestive and Liver Disease Unit, 2nd School of Medicine, University ‘La Sapienza’, Rome, Italy4 Medical Oncology Unit, University Hospital of Parma, Parma, Italy
(Correspondence should be addressed to G Rindi, Dipartimento di Patologia e Medicina di Laboratorio, Sezione Anatomia Patologica, Università degli Studi, via Gramsci, 14, I-43100 Parma, Italy; Email: guido.rindi{at}unipr.it)
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Abstract |
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionDeclaration of interestReferences |
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Generally, APC and β-catenin mutations are mutually exclusive in cancer, but β-catenin mutations are more frequent in small adenomas than in large or invasive colon cancers (Samowitz et al. 1999). This suggests that APC mutations confer greater selective advantage than β-catenin mutations, likely due to tumour suppressor functions of APC other than the mere control of the Wnt pathway. Indeed, APC is a multifunctional protein with roles in cell migration and adhesion (dependent or not of β-catenin/E-cadherin system and Wnt pathway), chromosome segregation, spindle assembly, apoptosis and neuronal differentiation (Hanson & Miller 2005). Mutations of the APC gene are quite rare in cancers outside the gut, while β-catenin gene mutations are common in desmoid, gastric cancer, hepatocarcinoma, medulloblastoma, melanoma, ovarian cancer, pancreatic cancer and prostate cancer (Kikuchi 2003).
In digestive neuroendocrine tumours, abnormal β-catenin expression was frequently found, though mutation of β-catenin exon 3 was reported by some and not by others, most often in the absence of APC mutations (Gerdes et al. 1999, Semba et al. 2000, Fujimori et al. 2001, Barshack et al. 2002, Li et al. 2002, Hervieu et al. 2006, Su et al. 2006). Additionally, APC promoter methylation was reported in digestive endocrine tumours (House et al. 2003, Arnold et al. 2004a).
The aim of this work is to assess the status of major players involved in Wnt pathway in a series of digestive endocrine tumours, investigating with a multimodal approach: i) APC and E-cadherin promoter methylation; ii) APC chromosomal locus alterations; iii) β-catenin and E-cadherin expression and iv) β-catenin and APC mutation.
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Methods |
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This study was approved by the ethic committee of the University of Parma and informed consent was obtained from all patients and/or guardians. Sixty benign and malignant endocrine tumours of the gastroenteropancreatic (GEP) tract from 59 patients were routinely formalin-fixed, paraffin-embedded and classified according to WHO criteria, with histology grade and stage as recently proposed (Table 1; Solcia et al. 2000, Pizzi et al. 2005, Rindi et al. 2006, 2007).
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DNA from tumours and normal adjacent mucosa were isolated by manual microdissection, resulting in 80% tumour cell enrichment as described (D'Adda et al. 2002).
Methylation
DNA methylation status of the CpG islands of APC and E-cadherin gene promoters were determined by chemical modification of genomic DNA with sodium bisulphite and subsequent methylation-specific PCR (MSP) as described (Pizzi et al. 2005). Primer sequences and annealing temperatures for amplification of methylated (m) and unmethylated (u) alleles of APC and E-cadherin are as previously reported (Herman et al. 1996, Tsuchiya et al. 2000).
Loss of heterozygosity (LOH) analysis
The highly polymorphic microsatellite marker D5S346 in 5q22-23 on the APC locus (Zauber et al. 2003) was investigated with primer sequences and amplification conditions (http://www.gdb.org) with fluorescently labelled 5'-primers (WellRed dyes, Research Genetics, Huntsville, AL, USA) as previously described (D'Adda et al. 2002). PCR products were analyzed by the CEQ 2000XL DNA analysis systems (Beckman Coulter Inc., Fullerton, CA, USA). The following ratio was calculated on the basis of peak height data: (lower allele/higher allele)tum/(lower allele/higher allele)norm. The values
0.6 (allelic imbalance
40%) were considered as indicative of LOH.
Fractional allelic loss (FAL) index calculation
Most of the samples here investigated have been previously characterized for LOH at the following microsatellite markers: PYGM, D11S4946 and D11S913 (D'Adda et al. 1999a, 2002, Pizzi et al. 2003), DXS989, DXS1100 and DXS1192 (D'Adda et al. 1999b, Pizzi et al. 2002), TP53, D3S1478, D3S1481, D18S58 and D18S61 (Pizzi et al. 2003), D3S1100 and D3S1621 (Pizzi et al. 2005) and D9S157 and D9S171 (manuscript in preparation). A mean FAL index was calculated from the ratio: (number of markers with LOH)/(total number of informative markers).
Immunohistochemistry (IHC)
General neuroendocrine markers, hormones and Ki-67 were investigated as described (Bordi et al. 1991). β-catenin and E-cadherin IHC was performed after thermal antigen retrieval using the mouse clones β-catenin-1 (1:400 Dako, Glostrup, Denmark) and HECD-1 (1:100 Zymed Laboratories, Inc., San Francisco, CA, USA). The cells of normal epithelial tissue were used as positive controls. Negative controls consisted of omission of the primary antibody. The fraction of tumour cells expressing β-catenin and/or E-cadherin was assessed, as well as the staining distribution (membranous, cytoplasmic and nuclear) according to Aust et al. (2001). Areas with stronger protein expression were evaluated in tumours with zonal staining pattern.
β-Catenin and APC gene mutation analysis
Samples showing altered β-catenin expression were further analysed for mutations in exon 3 of the β-catenin gene and in the mutation cluster region (MCR) of exon 15 of the APC gene (approximately codons 1300–1500 (Miyoshi et al. 1992)). The primers used for β-catenin gene were: CAT3F 5'-ATGGAACCAGACAGAAAAGC-3' and CAT3R 5'-GCTACTTGTTCTGAGTGAAG-3' (fragment size: 200 bp; Gerdes et al. 1999). APC mutation analysis was performed using four sets of primers, amplifying two overlapping portions of exon 15: 1) APCGn 5'-AAGAAACAATACAGACTTATTGT-3' and APC-Gc 5'-ATGAGTGGGGTCTCCTGAAC-3' (fragment G: codons 1256–1381, 377 bp; Doglioni et al. 2003); 2) APC-Hn 5'-ATCTCCCTCCAAAAGTGGTGC-3' and APC-Hc 5'-TCCATCTGGAGTACTTTCCGT G-3' (fragment H: codons 1359–1499, 421 bp; Doglioni et al. 2003); 3) APC-1f 5'-CATCAGCTGAAGATGAAATAGGA-3' and APC-1r 5'-GCAATCGAACGACTCTCAAA-3' (fragment APC-1: codons 1281–1402, 364 bp; Su et al. 2006) and 4) APC-2f 5'-ATGTTCAGGAGACCCCACTC-3' and APC-2r 5'-CACTCAGGCTGGATGAACAA-3' (fragment APC-2: codons 1376–1508, 396 bp; Su et al. 2006). PCR was performed in a final volume of 50 µl, with 25 pmol of each primer, 0.8 mM total dNTPs, 1.5 mM MgCl2 and 1.25 units AmpliTaq Gold (Applied Biosystem, Foster City, CA, USA). Conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 45 s, annealing at 58 °C (for CAT3, APC-1 and APC-2 primers) or 48 °C (for G and H fragments) for 45 s and elongation at 72 °C for 45 s. DNA sequencing was performed using Eurofins MWG Operon/M-Medical (Milano, Italy). Sequencing results were verified in our laboratory in both sense and antisense directions using DNA STAR PC software (Lasergene, Madison, WI, USA). Mutations were determined through alignment with normal sequences as reported in NCBI/Blast Human Genome database (http://blast.ncbi.nlm.nih.gov/Blast.cgi: ref|NT_034772.5|Hs5_34934 for APC, ref|NT_022517.17|Hs3_22673 for β-catenin). Mutations were named according to the nomenclature proposed by the Human Genome Variation Society (http://www.hgvsorg/) and submitted to the Human Gene Mutation Database, Cardiff (http://www.hgmd.cf.ac.uk).
Statistical analysis
The frequencies of specific gene alterations in different tumour groups were analyzed using two-tailed Fisher's exact test. Overall, survival curves were calculated using the method of Kaplan & Meier (1958). The log-rank test (Mantel 1966) was used to compare survival distributions. P<0.05 were considered significant in all analysis. SPSS software (version 8.0, SPSS Inc., Chicago, IL, USA) was used in all analyses.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionDeclaration of interestReferences |
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APC promoter methylation (as an example see Fig. 1A) was detected in 21 out of 59 (36%) of all cases and restricted to gastroduodenal tumours (75 and 60% of cases respectively; Table 2 and Fig. 2A, left). APC methylation was more frequent in poorly differentiated endocrine carcinomas (PDECs, occurring in 100% of gastric cases) than in well-differentiated neoplasms, either benign or malignant (P=0.042), in male than female patients (P=0.015), with no significant correlation with age (Table 3). Though not correlating with size, the presence of metastases nor mitotic count, APC promoter methylation strongly correlated with Ki67 index >2% (P<0.001), high histology grade (P<0.02) and stage (P=0.047), and poor outcome, being more frequent in patients dead of disease than in patients alive with or without evidence of disease (P=0.026) and barely missing statistical significance in survival analysis (median OS 100 vs 84 months, P=0.097; Fig. 2A, right and Table 3). APC methylation strongly correlated with chromosomal instability, as indicated by higher FAL index (0.3) in methylated tumours versus unmethylated (P<0.001, mean value 0.49±0.27 vs 0.18±0.26; Fig. 2B, left). In turn, FAL values 0.3 correlated with shorter survival (median OS: 24 months versus median not reached, P=0.017; Fig. 2B, right).
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APC locus marker LOH (as an example see Fig. 1B) was found in 14 out of 53 (26%) informative cases, with similar distribution as for APC methylation, i.e. in gastroduodenal tumours plus colon PDECs and one rectal well-differentiated endocrine tumour (WDET; Fig. 2A, left and Table 2). APC LOH was significantly more frequent in PDECs than in well-differentiated neoplasms (P=0.002) and in tumours with high histology grade (P=0.028) and high FAL index (P=0.001; Table 3).
Nuclear accumulation of β-catenin correlates with tumour aggressiveness and APC alterations
Disruption of normal membranous pattern (Fig. 2C, left), including various combinations of cytoplasmic accumulation and nuclear migration, occurred in the majority of GEP endocrine neoplasms (41 out of 54, 76%), independently of type and site (Tables 2 and 3). Nuclear accumulation (Fig. 2C, left) was found in 12 out of 54 cases (22%) co-segregating with aggressiveness. It was significantly more frequent in well-differentiated carcinomas (WDEC) than WDET (P=0.011) and in PDECs than well-differentiated neoplasms (P=0.033), correlating with high size (2 cm; P=0.002), the presence of metastases (P<0.001), mitotic count (P=0.011), Ki67 index (>2; P=0.011), high histology grade (P=0.005) and stage (P<0.001), tumour-related death (P=0.018) and shorter survival (median OS: 13 months versus median not reached, P=0.006, Fig. 2C, right). Finally, any APC alteration (methylation and/or LOH) correlated with β-catenin nuclear expression (P=0.047).
E-cadherin abnormality does not correlate with clinical-pathological features
E-cadherin promoter methylation was found in 2 out of 52 analyzed cases only (Table 2). The loss of normal membranous staining pattern and concurrent cytoplasmic accumulation was found in 18 out of 43 cases (42%; Table 2). E-cadherin alteration was more frequent in pancreatic tumours than in other neoplasms (6 out of 8 cases, 75%, P=0.044), in the absence of statistically significant correlation with any clinical-pathological feature or any other alteration investigated here.
Mutations in APC but not in β-catenin gene are found in tumours with altered β-catenin expression
Tumours with altered β-catenin expression (N=41) were analysed for mutations in exon 3 of β-catenin gene and exon 15 MCR of APC gene. No mutation was found in β-catenin gene (0 out of 40). Of 35 cases, 7 (20%) showed mutations in the APC gene (as an example see Fig. 1C), 2 of which in PDECs and 5 in well-differentiated neoplasms (Table 4). Most mutations (4 out of 7) were found in the midgut (4 out of 11 cases investigated for, 36%). One mutation was a 2 bp frameshift deletion causing a stop codon, as described in the literature (Miyoshi et al. 1992). All other alterations were single-base substitutions, missense mutations, one of which previously reported (Saito et al. 2002). Additionally, two single nucleotide polymorphisms (SNP) were identified: 1493ACG>ACA (T1493T) in seven cases and 1478ACG>AGA (R1478R) in one case. Since located at the very end of the MCR, assessment of 1493 SNP was possible in 14 cases only.
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Significance of multiple events cooperating in APC inactivation
A complete set of information on multiple/concurrent events of APC gene inactivation was available for 33 cases only, 32 of which with abnormal β-catenin expression (one undetermined; Table 5). In this series, a subset of nine cases displayed a double event (two hits), six with promoter methylation and LOH (all gastric) and three with other combinations. Overall, two hits identified a subset of aggressive cases with significant correlation with tumour differentiation (P=0.004), size (P=0.027), the presence of metastases (P=0.039), Ki67 (P=0.013), high grade (P=0.01) and stage (P=0.04), tumour-related death (0.018), high FAL (0.013) and nuclear β-catenin (P=0.011).
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Discussion |
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Mutational activation of Wnt signalling plays a key role in regulating intestinal epithelium fate and invariably initiates colorectal cancer, but it is also implicated in many other types of human tumours (Kikuchi 2003). The hallmark of Wnt pathway alterations is the stabilization of β-catenin, its migration in the nucleus where it determines the expression of various genes, involved in tumourigenesis including c-Myc and cyclin D1.
Data on Wnt pathway in GEP endocrine tumours are controversial. Some report suggest no role or some only as late event, others support its involvement although with limited role for APC (Fujimori et al. 2001, Hervieu et al. 2006). In specific, different frequencies of β-catenin mutations were reported in different series (Semba et al. 2000, Fujimori et al. 2001, Su et al. 2006). Nonetheless, abnormal β-catenin expression was frequently observed, often with concurrent loss of membranous E-cadherin expression, and usually associated with malignant features (Fujimori et al. 2001). The recent demonstration of APC promoter methylation in colorectal cancer and GEP endocrine tumours suggested an alternative mechanism for APC inactivation (Arnold et al. 2004a,b).
Here, we report frequent β-catenin alteration, with accumulation in the cytoplasm and/or nucleus, in GEP endocrine tumours (76% of cases), though the nuclear expression pattern only (22% of cases) correlated with biological parameters and co-segregated with aggressiveness. The nuclear localization of β-catenin was more frequent in PDECs than well-differentiated neoplasms and correlated with tumour size, mitotic count, Ki-67 index, grade, stage, presence of metastases, tumour-related death and shorter survival. Additionally, no β-catenin exon 3 mutation was observed in cases with altered β-catenin expression (N=40), suggesting alternative mechanism(s) for Wnt pathway activation. In contrast to previous studies (Semba et al. 2000, Fujimori et al. 2001, Li et al. 2002), we did not find statistically significant correlation between E-cadherin alterations and β-catenin expression and/or clinico-pathological features. The lack of correlation with E-cadherin alteration may find a possible explanation by the alternative role proposed for different β-catenin expression patterns (Doucas et al. 2005). The simple reduction or loss of membrane β-catenin indicates an alteration of the cell adhesion system through the cadherin pathway. By converse, the nuclear accumulation and concurrent increased cytoplasmic expression, as observed in our series, point to a transcriptional role for β-catenin in the Wnt pathway. Of the classical downstream transcriptional targets of β-catenin, cyclin D1 expression was previously investigated in the same series (Pizzi et al. 2005), proving not to correlate with β-catenin nuclear expression in a present analysis (unpublished). Alternative transcriptional targets of β-catenin should be sought for in endocrine cancer disease.
In our series, APC promoter methylation was almost restricted to gastroduodenal (36%) endocrine tumours, whereas it was consistently absent in pancreatic, ileal and colorectal neoplasms. This finding appears not to be part of the so-called CpG island methylation phenotype, since about 50% (11 out of 21) of cases with APC promoter methylation tested for showed no methylation for other genes (Pizzi et al. 2005 and unpublished). LOH at the APC locus in 5q21 largely reflected a similar distribution, with the exception of colonic PDECs and one rectal WDET. In the whole series, the presence of APC methylation and/or LOH correlated with β-catenin nuclear expression, indicating a central role for APC in Wnt pathway abnormality in gut endocrine tumours. Mutation of APC MCR, though conducted only in a subset of cases with altered β-catenin expression (N=35), was found in seven cases (20%). Mutations were frequently found in PDECs (2 out of 7) and midgut well-differentiated neoplasms (4 out of 11). The only other mutation was observed in a gastric WDEC in multiple endocrine neoplasia syndrome type 1 (MEN1). The possible association between FAP and MEN1 has been previously proposed (Sakai et al. 2002). Additionally, the 1493ACG>ACA SNP was often observed, its potential significance requiring further investigation.
The mechanism by which Wnt signalling is altered differs in distinct subset of GEP endocrine tumours, as shown by the restriction of APC promoter methylation to gastroduodenal tumours and frequent APC gene mutations in midgut neoplasms. These data provide further support to the hypothesis of different genetic background for different site endocrine tumours, as previously suggested (Rindi & Bordi 2003).
In our series, APC promoter methylation and/or LOH significantly correlated with various malignancy parameters including grade, stage and tumour death. In addition, in the subset of cases investigated for molecular events cooperating in APC inactivation, the combination of multiple hits resulted in stronger statistical association. Moreover, APC alterations strongly correlated with tumour mean FAL values, an index of chromosomal instability, in turn, strikingly correlating with shorter patient survival. The correlation between malignancy and chromosomal instability is a well-known phenomenon at least in pancreatic endocrine neoplasms (Rindi & Bordi 2003, Jonkers et al. 2007). Our data suggest a role for APC in the control of chromosome segregation in digestive endocrine cancer, as demonstrated in colorectal adenocarcinoma (Hanson & Miller 2005). Finally, the present findings support the effectiveness of the recently proposed histology grading and staging for endocrine tumours (Rindi et al. 2006, 2007).
In conclusion, in spite of methodology limits (namely the lack of direct demonstration of APC loss of expression), our data are proof of concept that Wnt pathway is often abnormal in aggressive GEP endocrine tumours, and this goes along with malignancy and survival. APC plays a major role in Wnt pathway activation, but the mechanisms of alteration differ depending on the anatomical site of origin: in gastroduodenal endocrine tumours via APC promoter methylation and in midgut through APC mutation. Additionally, APC alterations appear to play a central role in determining the high chromosomal instability in aggressive carcinomas.
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Declaration of interest |
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Acknowledgements |
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References |
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