Do Collision Tumors of the Gastroesophageal

Transcription

1 Collision tumors of the GEJ - Early-Onset Gastric Cancer - Milne et al 155 Do Collision Tumors of the Gastroesophageal Junction Exist? A Molecular Analysis Anya N.A. Milne 1, Ralph Carvalho, 1 Bas P. van Rees, 1 Jan J.B. van Lanschot, 2 G. Johan A. Offerhaus, 1 Marian A.J. Weterman. 1 1 Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands 2 Department of Surgery, Academic Medical Center, Amsterdam, The Netherlands American Journal of Surgical Pathology 2004 Nov;28():

2 156 Collision tumors of the GEJ - Early-Onset Gastric Cancer - Milne et al Abstract Collision tumors are thought to arise from the accidental meeting of two independent tumors. Here we present five gastro-esophageal junction tumors consisting of two collision tumors and three composite tumors (characterized by two divergent lineages originating from the same neoplastic clonal proliferation), as diagnosed on histology. In an attempt to prove this distinction at a genetic level we performed TP53 sequence analysis and p53 immunohistochemistry. In addition, loss of heterozygosity (LOH) analysis using ten microsatellite markers was carried out. An identical TP53 mutation and a similar pattern of retention and LOH were found in both neoplastic components of the presumed collision tumors, suggesting that both components are derived from a single precursor cell that undergoes divergent differentiation in the evolution of the tumor. In the composite group, one case had a genetic basis for the possible diagnosis of a collision tumor, with a TP53 mutation in the adenocarcinoma component only, and a different pattern of retention and loss of heterozygosity. These findings imply that it is not possible to recognize true collision tumors from immunohistological appearance alone and suggest that the longstanding histological criteria for the diagnosis of these neoplasms have no molecular basis.

3 Collision tumors of the GEJ - Early-Onset Gastric Cancer - Milne et al 157 Introduction Gastric and esophageal carcinomas remain a leading cause of cancer mortality worldwide. Moreover, the incidence of adenocarcinoma arising at the gastro-esophageal junction (GEJ) appears to be increasing. 17 These junctional adenocarcinomas may develop from a Barrett esophagus and Barrett carcinoma is currently the most rapidly increasing cancer in the Western world. 22 Since 1919 collision tumors were thought to arise from the accidental meeting and interpenetration of two independent tumors. 20 In 1961, the first case of a cardia collision carcinoma was described and the following three criteria were proposed: 5 1) the two components should show at least a partial topographical separation; 2) the squamous component should lie on the esophageal side of the tumor and the adenocarcinoma component on the gastric side; 3) there should be little or no evidence of intermediate histological structure. Others believed that an intermediate pattern at the point of collision was still acceptable for the diagnosis of a collision tumor. 24,31 Collision tumors need to be distinguished from composite tumors (characterized by two divergent lineages originating from the same neoplastic clonal proliferation), as it is possible that a different treatment is warranted, depending on the type of collision tumor encountered. Collision tumors occur at a higher frequency than would be expected statistically if their combined presence were merely coincidental. A possible explanation for this is the presence of field cancerization, which occurs due to long term exposure to carcinogens, whereby multiple transforming events give rise to genetically unrelated secondary primary tumors with independent mutations 2,6,23,25 and thus the chance that these tumors may in some instances collide is increased. In some situations environmental triggers explain the collision phenomenon, such as the association of Helicobacter pylori with the synchronous occurrence of MALT (mucosa-associated lymphoid tissue) lymphoma and adenocarcinoma of the stomach, 21,32 where a single carcinogenic agent interacts with the two neighboring tissues, inducing the development of tumors of different histogenesis in the same organ. 18 The collision of carcinomas with carcinoid tumors has also been reported frequently and it has been suggested that carcinoids can produce substances with a growth promoting effect accounting for the occurrence of a second tumor in the vicinity. 3 However, many collision tumors have no explanation for their occurrence, and as most diagnoses have been made on histology alone, the question exists as to whether histological classifications accurately reflect the molecular findings in these tumors. Genetic analysis would provide evidence regarding whether the tumors originate from the same or different clones. Here we present two tumors of the gastro-esophageal junction, which had the histological appearance of true collision tumors (Figure 1). In case A an adenocarcinoma collided with a squamous cell carcinoma, and in case B an adenocarcinoma (with a signet ring cell appearance) collided with a squamous cell carcinoma. We compared these two cases with three cases (C, D, and E) that had a histological appearance of an adenosquamous (composite) tumor. The finding of a tumor with glandular and squamous elements (adenosquamous carcinoma) is a well-recognized entity, included in the WHO classification 12 and has been characterized at a molecular level, 30 thus providing a reference point from which to compare the two collision tumors. In an effort to determine the clonality of the two separate components in each case, we characterized the molecular-genetic alterations of each tumor component including TP53 sequence analysis, loss of heterozygosity at ten chromosomal loci, and p53 immunohistochemistry.

4 158 Collision tumors of the GEJ - Early-Onset Gastric Cancer - Milne et al Patients and Methods This research was carried out in accordance with the ethical guidelines of the research review committee of the Academic Medical Center, Amsterdam. Description of Cases Case A: Macroscopically, a 9 cm diameter ulcerated tumor was present at the cardia, extending into the esophagus. Microscopically the tumor was composed of an adenocarcinoma adjacent to a squamous cell carcinoma with no intermingling at their interface (Figure 1A). A fibrous band separated the two components. Case B: Macroscopically a poorly circumscribed tumor 5 cm in maximum dimension was present in the distal esophagus extending through the gastro-esophageal junction into the cardia. Microscopically it consisted of a squamous cell carcinoma proximally and a diffuse signet ring cell carcinoma distally (Figure 1B). A thin fibrous band separated these components and no intermingling was observed. These findings of a tumor with diffuse signet ring cell and squamous cell carcinoma components is an entity that to our knowledge has never been described at this location. Case C: Macroscopically, a 3 cm diameter tumor was present at the gastro-esophageal junction, which was microscopically composed of distinct adenocarcinoma and squamous cell carcinoma components. There was widespread intermingling of components. Case D: A polypoid tumor 10 cm in length was present at the gastro-esophageal junction, which consisted of a squamous cell carcinoma extending through the muscularis propria. Near the surface a well-differentiated adenocarcinoma component was present with some intermingling with the adjacent squamous cell component. Case E: A 1.5 cm diameter tumor was present at the gastro-esophageal junction. Microscopically the majority of the tumor consisted of a poorly differentiated tumor with distinct separate adenocarcinoma and squamous cell carcinoma areas intermingled with the poorly differentiated areas. Based on histology, a collision tumor was diagnosed in cases A and B, and an adenosquamous (composite) tumor in cases C, D, and E. Figure 1 Haematoxylin and eosin staining of cases A (x50) and B (x100) showing the interface of the adenocarcinoma and squamous cell carcinoma components. (See page 202 for colour figure)

5 Collision tumors of the GEJ - Early-Onset Gastric Cancer - Milne et al 159 Table 1 Microsatellite markers used for loss of heterozygosity analysis Microsatellite Marker Chromosomal Region Tumour Suppressor Gene D3S1478 3p21 FHIT; hmlh1 D5S346 5q21 APC D9S171 9p21 p14 ARF ; p16 INK4A /CDKN2A; p15 INK4B /CDKN2B D10S q23 PTEN D14S68 14q24.3 frequently deleted region in Barrett carcinomas D16S q22.1 CDH1 D18S64 18q21.32 DCC; DPC4/SMAD4; SMAD2 TP53 17p13.1 TP53 P53Alu 17p13.1 TP53 Bat26 2p16 MSI consensus marker Immunohistochemistry Immunohistochemistry for p53 was performed using the monoclonal antibody combination of DO-7 and BP53-12 (Neomarkers, Union City, CA, USA). Sections were deparaffinized and antigen retrieval was carried out by 10 minutes of boiling in 10 mmtris/1 mm EDTA (ph 9). Subsequently slides were immersed in 0.3% hydrogen peroxide in methanol for 30 minutes and non-specific binding was blocked with 5% normal goat serum for 1 hour at room temperature. The sections were incubated with the primary antibody at a 1:2000 dilution for 1 hour at room temperature. The Ultravision antipolyvalent HRP detection system (Lab Vision Corp., Fremont, CA, USA) was used to visualize antibody binding sites with 3,3 -diaminobenzidine as a chromogen. Sections were counterstained with haematoxylin and p53 immunostaining was considered positive when staining was present in >30% of tumor cells. TP53 Mutation Analysis The TP53 gene was initially amplified from RT-PCR material derived from fresh frozen tissue of one neoplastic component of each tumor. Mutations were subsequently confirmed in both tumor components using DNA derived from paraffin-embedded material. Due to unavailability of frozen material in case C all exons were amplified using DNA isolated from paraffin embedded material. DNA Extraction and Microdissection In case A, the squamous cell carcinoma component was composed of squamous islands surrounded by a dense lymphocytic infiltrate, thus requiring microdissection with the PALM Laser Microbeam Microdissection system (Microlaser Technologies, Bernried, Germany). Slides for this method were prepared by fixing PALM LPC membrane onto specialized thin glass slides, applying 0.1% poly-l-lysine and mounting a 6μm tissue section. This was subsequently stretched on a hot plate at 50ºC and left at 60ºC for 1 hour. For the remaining 4 cases, serial 6 μm sections were cut with a microtome, deparaffinized using a standard xylene and serial ethanol protocol, stained with haematoxylin and carefully microdissected using an inverted microscope and a sterile needle. Both cases had discrete areas of each tumor thus facilitating accurate microdissection without risk of contamination with normal tissue. Normal control tissue was dissected from lymph nodes or, in the case where lymph node metastases occurred, from distant normal gastric mucosa. DNA was isolated using the PUREGENE DNA Isolation Kit (Gentra Systems, Minneapolis, USA) according to manufacturer s instructions. DNA

6 160 Collision tumors of the GEJ - Early-Onset Gastric Cancer - Milne et al concentrations were measured using PicoGreen dsdna Quantitation Kit (Molecular Probes, Leiden, The Netherlands). RT-PCR Twenty 4-5 μm thick sections of frozen material containing >90% tumor cells were used for RNA extraction using Trizol (Invitrogen, Breda, The Netherlands) and the quality of RNA was assessed by agarose gel electrophoresis. Subsequently, complementary DNA was synthesized using the M- MLV RT enzyme kit (Invitrogen), pd (N)6 random primers (Amersham Biosciences, Roosendaal, The Netherlands) and RNA inhibitor (Roche, Basel, Switzerland), according to the manufacturer s instructions. PCR amplification was performed to include the entire TP53 coding sequence by amplification of three overlapping segments, using 1 μl of cdna (total 30 μl) and the following primers: 5 - GCTTTCCACGACGGTGACA-3 (A forward), 5 -ACTCCCCTGCCCTCAACAA-3 (A reverse), 5 -TCTGTCCCTTCC-3 (B forward), 5 -GCTCTGACTGTACCACCATC- 3 (B reverse), 5 -TTGCGTGTGGAGTATTTGGA-3 (C forward) and 5 - GGTCTTTGAACCCTTGCTTGC-3 (C reverse). 35 cycles were carried out using 0.2 mm dntps, 1.5 mm MgCl 2, 1 mm forward and reverse primers, at an annealing temperature of 55 C and 4 units of Platinum Taq DNA polymerase (Invitrogen) in a buffer supplied by the manufacturer, with an end volume of 50μl in a Peltier Thermal Cycler (DYAD ), (Biozyme, Blaenavon, South Wales, UK). PCR on Paraffin Material The following primers were used for amplification of paraffin embedded material: exon 2 and 3 forward: 5 -GCTTGGGTTGTGGTGAAACA-3, reverse: 5 -TCCAGGTCCCCAGCCCAAC- 3 ; exon 4 fragment 1 forward: 5 - ACCTGGTCCTCTGACTGC-3, reverse: 5 - TTTTCTGGGAAGGGACAG-3 ; fragment 2 forward: 5 -AAGCTCCCAGAATGCCAG- 3, reverse: 5 -AAGCCTAAGGGTGAAGAG-3 ; exon 5 forward: 5 - TGCTGCCGTGTTCCAGTTGC-3, reverse: 5 -AGGCCTGGGGACCCTGGGCAACC- 3 ; exon 6 forward: 5 -CTCTGATTCCTCACTGATTGCTCT-3, reverse 5 - GGAGGGCCACTGACAACCA-3 ; exon 7 forward: 5 -CTTGCCACAGGTCTCCCCAA- 3, reverse: 5 -AGGGGTCAGCGGCAAGCAGA-3 ; exon 8 forward: 5 - TGCCTCTTGCTTCTCTTTTC-3, reverse: 5 -TCCTCCACCGCTTCTTGTC-3 ; exon 9 forward: 5 -AGACCAAGGGTGCAGTTATG-3, reverse: 5 - CGGCATTTTGAGTGTTAGA-3 ; exon 10 forward: 5 -GTTGCTTTTGATCCGTCA-3, reverse: 5 -GAATCCTATGGCTTTCCAAC-3 ; exon forward: 5 -GGGCACAGACCCTCTCAC-3, reverse: 5 -GTGGGAGGCTGTCAGTGG-3. At least 10 ng of DNA and 10 pmol of primer was used for each reaction and an annealing temperature between 55 and 60º was used. Other conditions were identical to the PCR amplification described above. Sequence Analysis Following purification of PCR products with the QIA quick PCR purification kit (Qiagen, Leusden, The Netherlands), the sequences were analyzed using the ready reaction Big Dye Terminator Cycle Sequence kit (Applied Biosystems, Foster City, CA, USA) and an ABI 3100 automated sequencer (Applied Biosystems).

7 Collision tumors of the GEJ - Early-Onset Gastric Cancer - Milne et al 161 Loss of Heterozygosity LOH analysis was carried out using ten polymorphic microsatellite repeat markers. The sequences of the primers and their corresponding location were selected through the Genomic Data Base ( (Table 1). The primers used for the p53alu marker were 5 - GAATCCGGGAGGAGGTTG-3 (forward) and 5 -TTCTGCTCCTTTTTTGGCTG-3 (reverse) and those used for the TP53 marker were 5 -AGGGATACTATTCAGCCCGAGGTG- 3 (forward) and 5 - ACTGCCACTCCTTGCCCCATTC-3 (reverse). PCR amplification was performed using 35 cycles in a 20 μl reaction volume containing 0.2 mm dntps, 1.5 mm MgCl 2, 1.5 unit of Platinum Taq DNA polymerase in buffer supplied by the manufacturer, and 0.5mM of each primer, one of which was labeled with a fluorescent marker. Annealing temperatures ranging from 50ºC to 64ºC were used. A minimum of 10 ng of DNA was used for each PCR reaction to prevent induced losses. LOH analysis was carried out using an automated ABI 3100 sequencer with a Genescan 350ROX size standard (Applied Biosystems) and the manufacturer s Genescan 2.1 software. The ratio of peak heights of the two alleles for tumor sample and normal was calculated and subsequently the normal ratio was divided by the tumor ratio. A value below 0.6 or above 1.6 was interpreted as evidence of LOH whereas values between these figures were considered retention of heterozygosity. All losses were confirmed by at least one repeat reaction. Results P53 Abnormalities Due to their frequent and early occurrence in gastro-esophageal tumors, TP53 mutations provide a powerful molecular tool for assessment of clonality. Thus direct TP53 sequence analysis and immunohistochemistry for p53 was carried out for all cases (A-E). The results are presented in Table 2. All cases but one (case C) had a TP53 mutation. Both tumor components of the two presumed collision tumor cases (A and B) shared the same TP53 mutation. The group of composite tumors as diagnosed on histology consisted of cases C, D, and E. In case C a known polymorphism was found in both tumor components. Case D however had a TP53 mutation in the adenocarcinoma component only, with no mutation found in the squamous component. In case E the same TP53 mutation was found in both tumor components. Table 2 Results of TP53 sequence analysis and immunohistochemistry Case Histology Mutation Ad P53 IHC Mutation SC P53 IHC Position Amino Acid Change A B C D E collision tumor collision tumor composite tumor composite tumor composite tumor G to A positive G to A positive exon 8, codon 266 G to A positive G to A positive exon 8, codon 273 polymorphism negative polymorphism negative exon 4, codon 72 C to G positive none negative exon 8, codon 278 C to T positive C to T positive exon 9, codon 331 Gly to Glu Arg to Hist Arg to Pro Pro to Arg Gln to stop codon

8 162 Collision tumors of the GEJ - Early-Onset Gastric Cancer - Milne et al Table 3 Results of Loss of Heterozygosity Analysis Microsatellite Marker Case A Case B Case C Case D Case E SC AC SC AC SC AC SC AC SC AC D3S1478 LL R LL LL R R R R R R D5S346 LS LS LS LS LL LL LL R LS LS D9S171 R R R R R R LS R R R D10S2491 LS LL LL LL R R R LS R R D14S68 R R R R R R LL LL R R D16S2624 R R N.I. N.I. R R LL R R R D18S64 R LS LL LL LL LL N.I. N.I. LS LS TP53 LS LS LL LL LL LL LS LS LS LS P53Alu N.I. N.I. N.I. N.I. N.I. N.I. LL LL LS LS Bat26 No MSI No MSI No MSI No MSI No MSI No MSI No MSI No MSI No MSI No MSI LS Loss of small Allele; LL Loss of large Allele; AC Adenocarcinoma; SC Squamous Cell Carcinoma Immunohistochemistry for p53 corresponded with the molecular genetic findings and showed overexpression in both tumor components in cases A, B, and E. Case C was negative for both components and Case D showed positivity in the adenocarcinoma component only (figure 2). Detection of Loss of Heterozygosity Further evidence regarding the clonality of these tumors was obtained through LOH analysis of each component using a panel of ten polymorphic markers (Table 1), at sites near known or suspected tumor suppressor genes considered potentially important in gastro-esophageal carcinogenesis. The results are shown in Table 3. Both neoplastic components of the two presumed collision tumor cases (A and B) had a similar pattern of loss and retention. In Case A the results were the same for five loci and three markers revealed a difference between the neoplastic components. The results for Case B were identical in both components for all the markers studied. In all cases where LOH for both components was observed, this involved the same allele. One exception was at 10q23 in case A, where loss of the large allele was found in the adenocarcinoma component and a loss of the small allele in the squamous component. The results for the group of three presumed composite tumors revealed an identical pattern of loss and retention for cases C and E. Case D however showed a different pattern of retention and loss at four of the eight informative loci. Discussion The pathogenesis of human cancers is thought to be the result of a stepwise accumulation of genetic alterations involving oncogenes and tumor suppressor genes. A tumor can be considered clonal with respect to crucial early genetic events 7,15 whereas it is often heterogeneous for genetic changes occurring late in tumor progression. Molecular genetic analysis provides stronger evidence for a multi- or monoclonal origin than phenotypic appearance, as important genetic alterations will be retained during carcinogenesis whereas phenotypic features may change. If two tumors arise

9 Collision tumors of the GEJ - Early-Onset Gastric Cancer - Milne et al 163 independently and are associated by coincidence only, the genetic alterations present are expected to be different from each other because of the different tumor origins. Mutational analysis of a molecule critical to carcinogenesis is central in providing clues regarding clonality. 2,6 In gastro-esophageal tumors, the prevalence, early occurrence and wide variety of TP53 mutations distributed throughout the gene, make sequence analysis of TP53 a useful tool for differentiating between biclonal and monoclonal origins. Of note, even in the situation of field cancerization of the esophagus, where G to T transitions of TP53 can occur throughout the esophagus due to environmental factors such as tobacco, the resulting mutations are not specific but are dispersed throughout numerous codons. 13 Thus in field cancerization a wide range of different TP53 mutations occurring at different times are present within the affected field which give rise to the second primary tumors characteristic of field cancerization. This is in contrast to the role of aflotoxin in hepatocellular carcinoma where it induces a specific TP53 mutation at codon 249. Aflotoxin is one of the only known carcinogens to induce a specific TP53 mutation in human cancer, with no such specific mutations described in the esophagus. 13 It is also possible to follow the clonal evolution of cancer with markers that identify LOH. 4,10 In this study, a molecular genetic approach using TP53 sequence analysis with supporting LOH analysis was applied to investigate five gastro-esophageal junction tumors, each of which consisted of two histologically separate components. Two of these tumors (A and B) had the histological appearance of collision tumors and the other three cases (C, D and E) were deemed composite tumors on histology. Surprisingly, the molecular data provided here showed no evidence that case A and B were collision tumors, despite their characteristic histological appearance. In both cases, the tumor components had an identical TP53 mutation and so the likelihood that they are collision tumors is virtually zero. This conclusion is supported by the concordant LOH at multiple loci, again providing evidence that the two neoplastic components initially arose from a common precursor. The results of the group of presumed composite tumors also revealed interesting results. Cases C and E had an identical pattern of loss and retention on LOH analysis thus confirming their composite nature. In case D however, a TP53 mutation was found in the adenocarcinoma component only, with no TP53 mutation found in the squamous component. The LOH findings of the two components were also disparate with a different pattern of results at four of the eight markers. These data provide evidence suggesting that this case may be a true collision tumor. However, it can be argued that this TP53 mutation could have occurred as a late event and that the two tumor components may share early genetic events and thus have the same clonal origin. The difficulty of distinguishing a clonal origin with subsequent sub-clone formation from genetically unrelated tumors with widespread instability is highlighted in this instance. The results show that despite a striking difference in morphology and relative separation of tumor components, a collision tumor cannot be assumed on appearance alone. There is no definitive explanation for the unusual appearance of these neoplasms but it is likely that growth factors have a strong influence on morphology and that the different microenvironments of the esophagus and cardia exert different effects on the two tumor components. Heterogeneity within a tumor often occurs as a result of increasing genetic instability during tumor progression and the accumulation of different molecular and genetic aberrations may also contribute to a difference in morphology. An example of tumor heterogeneity can be seen in case A at loci 3p21 and 18q21.32 where the differences in LOH may represent a diversion in tumor progression and formation of sub-clones. At 10q23 in case A, the pattern of LOH was unusual in that the small allele was lost in one component and the large allele in the other component. It is possible that these findings do not actually represent a real physical loss of this marker but rather have occurred due to an

10 164 Collision tumors of the GEJ - Early-Onset Gastric Cancer - Milne et al aberrant mitotic crossing over event resulting in two homozygous daughter cells, thus giving the appearance of a loss of heterozygosity. In many of the cases studied (B, C, E) the pattern of LOH was identical for both components. However, genetic differences accounting for the appearance of these components may exist at loci other than those examined here. To our knowledge, this is the first study seeking molecular genetic evidence for the histological diagnosis of collision tumors through both mutation analysis and LOH analysis. The literature regarding collision tumors consists mainly of case reports, most of which are based on histological appearance and immunohistochemistry. Given the results of cases A and B in this study, many of the collision tumors reported in the literature may represent clonal neoplasms with two divergent phenotypes. In fact in examples where genetic analysis has been performed, such as in carcinosarcomas of the uterus, most cases have been found to be clonal, thus representing carcinomas with sarcomatous areas, i.e. composite tumors. 8,19,26 Similar findings have been found for other putative collision tumors: gynecological tumors, 1,16 tumors in the pancreas, 27 and some tumors with glandular and neuroendocrine components. 9 This suggests that collisions tumors have been widely over-diagnosed in the past. Despite this possible over-diagnosis of collision tumors there are reports where genetic techniques have been used to show genetic differences in collision tumors 14,29,10 demonstrating that collision tumors do indeed occur in various parts of the body. In the case of female patients (this study consists of males only) a clonality X-inactivation analysis based on methylation pattern is another useful method to demonstrate clonality. 28 Although histological examination is the cornerstone of our practice of pathology, the molecular-genetic era has provided the tools to resolve issues that until now have been theoretical debates. From this study we can surmise that it is not possible to recognize true collision tumors from histological appearance alone, and that the criteria for the diagnosis of these neoplasms does not always hold true. Indeed the occurrence of a collision tumor is probably far rarer than what is reflected in the literature. The finding of a genetic basis for clonality in the cases of presumed collision tumors and the finding of a possible collision tumor among presumed composite tumors challenges our methods of diagnosis of these tumors and suggests that the longstanding histological criteria for the diagnosis of these neoplasms have no molecular basis. Acknowledgements This study was supported by the Vanderes foundation. The authors thank Folkert Morsink and Alex Musler for their technical assistance and Rein Visser and Wilfred Meun for assistance with figures. References 1. Abeln EC, Smit VT, Wessels JW, et al. Molecular genetic evidence for the conversion hypothesis of the origin of malignant mixed mullerian tumours. J Pathol 1997; 183: Chung KY, Mukhopadhyay T, Kim J, et al. Discordant p53 gene mutations in primary head and neck cancers and corresponding second primary cancers of the upper aerodigestive tract. Cancer Res 1993; 53:

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