Publications by authors named "Folkert Morsink"

44 Publications

Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size.

Gut 2021 Apr 13. Epub 2021 Apr 13.

Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Objective: Recent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown.

Design: An international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS).

Results: ATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (p<0.001). The 5-year RFS rates for patients with ATRX/DAXX-negative and ALT-positive NF-PanNETs were 40% and 42% as compared with 85% and 86% for wild-type NF-PanNETs (p<0.001 and p<0.001). Shorter 5-year RFS rates for ≤2.0 cm NF-PanNETs patients were also seen with ATRX/DAXX loss (65% vs 92%, p=0.003) and ALT (60% vs 93%, p<0.001). By multivariate analysis, ATRX/DAXX and ALT status were independent prognostic factors for RFS. Conversely, classifying NF-PanNETs by ARX/PDX1 expression did not independently correlate with RFS. Except for 4% of pulmonary carcinoids, ATRX/DAXX loss and ALT were only identified in primary (25% and 29%) and NF-PanNET metastases (62% and 71%).

Conclusions: ATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary.
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http://dx.doi.org/10.1136/gutjnl-2020-322595DOI Listing
April 2021

Status of CHEK2 and p53 in patients with early-onset and conventional gastric cancer.

Oncol Lett 2021 May 3;21(5):348. Epub 2021 Mar 3.

Department of Human Anatomy, Medical University of Lublin, 20-090 Lublin, Poland.

Gastric cancer (GC) is the fourth most common cause of cancer-associated death. Based on the age at diagnosis, GC is divided into early-onset GC (EOGC; ≤45 years) and conventional GC (CGC; >45 years). Mutations in the cell cycle checkpoint kinase 2 () and genes are associated with several types of cancer; however, their genetic defects in GC remain poorly understood. The aim of the present study was to determine the subcellular distribution of the CHEK2 protein and its redistribution following DNA damage, to improve the understanding of the DNA damage response. Genetic alterations and patterns of expression of CHEK2 and p53 proteins were investigated to identify potential biological markers and indicators of GC development. Additionally, the affected signaling pathways and their clinical importance in GC development and associated syndromes were investigated. A total of 196 GC samples (89 CGC and 107 EOGC samples) were used in the present study. DNA from 53 samples (18 CGC and 35 EOGC samples) was sequenced using targeted next-generation sequencing technology to identify and compare common and rare mutations associated with GC. Subsequently, the cytoplasmic and nuclear expression levels of CHEK2, phosphorylated (p)-CHEK2 at threonine 68 and p53 in GC tissues were determined via immunohistochemistry. Sequencing resulted in the identification of 63 single nucleotide polymorphisms (SNPs) in the gene amongst 5 different variants, and the intron variant c.319+379A>G was the most common SNP. In the gene, 57 different alterations were detected amongst 9 variant types, and the missense variant c.215C>G was the most common. Nuclear CHEK2 expression was high in both the EOGC and CGC subtypes. However, the prevalence of cytoplasmic CHEK2 expression (P<0.001) and nuclear p-CHEK2 expression (P=0.011) was significantly higher in CGC compared with in EOGC tissues. There was a statistically significant difference between high and low cytoplasmic CHEK2 expression in patients with p53-positive EOGC compared with in patients with p53-positive CGC (P=0.002). The present study was designed to determine the association between CHEK2 and p53 expression patterns in patients with EOGC and CGC, as well as genetic alterations in the and genes.
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http://dx.doi.org/10.3892/ol.2021.12609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7967923PMC
May 2021

Genome Methylation Accurately Predicts Neuroendocrine Tumor Origin: An Online Tool.

Clin Cancer Res 2021 Mar 22;27(5):1341-1350. Epub 2020 Dec 22.

Department of Pathology, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.

Purpose: The primary origin of neuroendocrine tumor metastases can be difficult to determine by histopathology alone, but is critical for therapeutic decision making. DNA methylation-based profiling is now routinely used in the diagnostic workup of brain tumors. This has been enabled by the availability of cost-efficient array-based platforms. We have extended these efforts to augment histopathologic diagnosis in neuroendocrine tumors.

Experimental Design: Methylation data was compiled for 69 small intestinal, pulmonary, and pancreatic neuroendocrine tumors. These data were used to build a ridge regression calibrated random forest classification algorithm (neuroendocrine neoplasm identifier, NEN-ID). The model was validated during 3 × 3 nested cross-validation and tested in a local and an external cohort ( = 198 cases).

Results: NEN-ID predicted the origin of tumor samples with high accuracy (>95%). In addition, the diagnostic approach was determined to be robust across a range of possible confounding experimental parameters, such as tumor purity and array quality. A software infrastructure and online user interface were built to make the model available to the scientific community.

Conclusions: This DNA methylation-based prediction model can be used in the workup for patients with neuroendocrine tumors of unknown primary. To facilitate validation and clinical implementation, we provide a user-friendly, publicly available web-based version of NEN-ID.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3281DOI Listing
March 2021

Expression of cyclooxygenase-2 and mucin 1 in colorectal cancer.

Mol Clin Oncol 2020 Nov 20;13(5):52. Epub 2020 Aug 20.

Department of Surgical Oncology, Medical University of Lublin, Lublin 20-090, Poland.

In colorectal cancer (CRC), pathological factors that correlate with negative prognosis include, among others, overexpression of cyclooxygenase-2 (COX-2) and abundant expression of mucin 1 (MUC1). COX-2 overexpression may therefore be associated with MUC1 overexpression. The aim of the present study was to investigate the possible correlation between COX-2 and MUC1 expression and to assess the correlation between their individual expression and the clinicopathological features of patients, paying particular attention to survival. The following data was collected from the 170 patients with CRC included in the present study: Age, sex, tumour localization, disease stage and survival. Tumour samples were immunostained with antibodies against COX-2 and MUC1. Protein expression was scored, relative to reference staining, and correlated with the clinicopathological data of patients. The results revealed no correlation between the expressions of COX-2 and MUC1, or with any of the studied clinicopathological variables. In addition, the expression of the two proteins were not associated. Neither of the proteins demonstrated prognostic value for survival. The present study did not confirm a direct relationship between the expressions of COX-2 and MUC1, or between the expression of either protein and the clinicopathological features of patients, including survival.
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http://dx.doi.org/10.3892/mco.2020.2122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453394PMC
November 2020

The decisive role of molecular pathology in presumed somatic metastases of type II testicular germ cell tumors: report of 2 cases.

Diagn Pathol 2020 Jul 25;15(1):99. Epub 2020 Jul 25.

Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Molecular diagnostics can be decisive in the differential diagnosis between a somatic metastasis of type II testicular germ cell tumor (TGCT) or a second primary carcinoma. This is in line with recent recommendations from the International Society of Urological Pathology, based on an international survey which showed that molecular testing is currently only performed by a minority of urological pathologists.

Case Presentations: This case report illustrates the necessity of molecular testing in two patients with a history of type II TGCT and a metastatic (retro) peritoneal carcinoma years later. The genetic hallmark of type II TGCT, chromosome 12p gain, was studied by fluorescence in situ hybridization and whole genome methylation profiling in case 1, and by single nucleotide polymorphism (SNP)-array in case 2. Next generation sequencing (NGS) was used to further explore clonality between the primary TGCT and peritoneal metastasis in case 2. In case 1, chromosome 12p gain was found in the primary type II TGCT and in the acinar cell carcinoma of the metastatic malignancy. In case 2, SNP array showed 12p gain in the epithelial component of the primary teratomatous TGCT but not in the peritoneal adenocarcinoma. Furthermore, NGS showed no mutations in the primary teratomatous TGCT but a KRAS and GNAS mutation in the peritoneal adenocarcinoma, suggestive of an appendicular origin.

Conclusions: Without the molecular data, both cases would have been regarded as a metastatic TGCT with development of somatic-type malignancy, which appeared a wrong diagnosis for case 2. These cases demonstrate the importance of molecular methods as an adjunct in today's pathology practice.
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http://dx.doi.org/10.1186/s13000-020-01011-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382836PMC
July 2020

High-Throughput Sequencing of Gastric Cancer Patients: Unravelling Genetic Predispositions Towards an Early-Onset Subtype.

Cancers (Basel) 2020 Jul 21;12(7). Epub 2020 Jul 21.

Department of Human Anatomy, Medical University of Lublin, 20-090 Lublin, Poland.

Background: Gastric cancer is the fourth most common cause of cancer-related death. Currently, it is broadly accepted that the molecular complexity and heterogeneity of gastric cancer, both inter- and intra-tumor, display important barriers for finding specific biomarkers for the early detection and diagnosis of this malignancy. Early-onset gastric cancer is not as prevalent as conventional gastric carcinoma, but it is a preferable model for studying the genetic background, as young patients are less exposed to environmental factors, which influence cancer development.

Aim: The main objective of this study was to reveal age-dependent genotypic characteristics of gastric cancer subtypes, as well as conduct mutation profiling for the most frequent alterations in gastric cancer development, using targeted next-generation sequencing technology.

Patients And Methods: The study group included 53 patients, consisting of 18 patients with conventional gastric cancer and 35 with an early-onset subtype. The DNA of all index cases was used for next-generation sequencing, employing a panel of 94 genes and 284 single nucleotide polymorphisms (SNPs) (TruSight Cancer Panel, Illumina), which is characteristic for common and rare types of cancer.

Results: From among the 53 samples processed for sequencing, we were able to identify seven candidate genes (, and ) and nine variants among them: one splice_acceptor, four synonymous, and four missense variants. These were selected for the age-dependent differentiation of gastric cancer subtypes. We found four variants with C-Score ≥ 10, as 10% of the most deleterious substitutions: rs1800862 (), rs10138997 (), rs2230009 (), and rs2959656 (). We identified 36 different variants, among 24 different genes, which were the most frequent genetic alterations among study subjects. We found 16 different variants among the genes that were present in 100% of the total cohort: (rs2746462), (rs1670283), (rs2958057), (rs4925828; rs11342077, rs398010167; rs2721190), (rs326212), (rs540012), (rs4930199), (rs659243), (rs1169305), (rs206075; rs169547), (rs9514066; rs9514067), and (rs7183618).

Conclusions: The technology of next-generation sequencing is a useful tool for studying the development and progression of gastric carcinoma in a high-throughput way. Our study revealed that early-onset gastric cancer has a different mutation frequency profile in certain genes compared to conventional subtype.
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http://dx.doi.org/10.3390/cancers12071981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409326PMC
July 2020

Degree and site of chromosomal instability define its oncogenic potential.

Nat Commun 2020 03 20;11(1):1501. Epub 2020 Mar 20.

Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands.

Most human cancers are aneuploid, due to a chromosomal instability (CIN) phenotype. Despite being hallmarks of cancer, however, the roles of CIN and aneuploidy in tumor formation have not unequivocally emerged from animal studies and are thus still unclear. Using a conditional mouse model for diverse degrees of CIN, we find that a particular range is sufficient to drive very early onset spontaneous adenoma formation in the intestine. In mice predisposed to intestinal cancer (Apc), moderate CIN causes a remarkable increase in adenoma burden in the entire intestinal tract and especially in the distal colon, which resembles human disease. Strikingly, a higher level of CIN promotes adenoma formation in the distal colon even more than moderate CIN does, but has no effect in the small intestine. Our results thus show that CIN can be potently oncogenic, but that certain levels of CIN can have contrasting effects in distinct tissues.
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http://dx.doi.org/10.1038/s41467-020-15279-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083897PMC
March 2020

Alternative Lengthening of Telomeres and Differential Expression of Endocrine Transcription Factors Distinguish Metastatic and Non-metastatic Insulinomas.

Endocr Pathol 2020 Jun;31(2):108-118

Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.

Insulin-producing pancreatic neuroendocrine tumors (PanNETs)/insulinomas are generally considered to be indolent tumors with an excellent prognosis after complete resection. However, some insulinomas have a poor prognosis due to relapses and metastatic disease. Recently, studies in non-functional PanNETs indicated that behavior can be stratified according to alpha- and beta-cell differentiation, as defined by expression of the transcription factors ARX and PDX1, respectively. It is unknown whether similar mechanisms play a role in insulinomas. Therefore, we determined ARX and PDX1 expression in a cohort of 35 sporadic primary insulinomas and two liver metastases of inoperable primary insulinomas. In addition, WHO grade and loss of ATRX or DAXX were determined by immunohistochemistry, and alternative lengthening of telomeres (ALT) and CDKN2A status by fluorescence in situ hybridization. These findings were correlated with tumor characteristics and clinical follow-up data. In total, five out of 37 insulinoma patients developed metastatic disease. Metastatic insulinomas were all larger than 3 cm, whereas the indolent insulinomas were smaller (p value < 0.05). All three primary insulinomas that metastasized showed ARX expression, 2/3 showed ALT, and 1/3 had a homozygous deletion of CDKN2A as opposed to absence of ARX expression, ALT, or CDKN2A deletions in the 32 non-metastatic cases. The two liver metastases also showed ARX expression and ALT (2/2). The presence of ARX expression, which is usually absent in beta-cells, and genetic alterations not seen in indolent insulinomas strongly suggest a distinct tumorigenic mechanism in malignant insulinomas, with similarities to non-functional PanNETs. These observations may inform future follow-up strategies after insulinoma surgery.
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http://dx.doi.org/10.1007/s12022-020-09611-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250793PMC
June 2020

Assessment of ARX expression, a novel biomarker for metastatic risk in pancreatic neuroendocrine tumors, in endoscopic ultrasound fine-needle aspiration.

Diagn Cytopathol 2020 Apr 17;48(4):308-315. Epub 2019 Dec 17.

Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: The transcription factors ARX and PDX1, and alternative lengthening of telomeres (ALT) were recently described as prognostic markers for resected non-functional pancreatic neuroendocrine tumors (PanNETs). ALT positive tumors with ARX expression relapse most often. Currently, tumor size is the only preoperative marker used to decide whether or not to operate, thus additional preoperative prognostic markers are needed. Therefore, it is critical to assess the performance of these biomarkers on preoperative cytologic specimens.

Methods: Endoscopic fine-needle aspiration cellblock material and corresponding surgical specimens of 13 patients with PanNETs were assessed for histology, immunohistochemical staining of ARX, PDX1, Synaptophysin, Ki67, and telomere-specific fluorescence in situ hybridization to detect ALT, and then associated with clinicopathological features. Scoring for ARX and PDX1 was performed blinded by two independent observers.

Results: Of the 13 surgical specimens, 8 were ARX+/PDX1-, 2 ARX-/PDX1+, and 3 ARX+/PDX1+. Concordance between cytologic and surgical specimens for ARX protein expression was 100%, whereas concordance for PDX1, ALT, and WHO tumor grade was 85%, 91%, and 73%, respectively. There was a perfect inter-observer agreement in ARX and PDX1 scoring.

Conclusion: ARX can reliably be determined in cytologic specimens and has low inter-observer variability. For cytology, false-positive PDX1 expression was observed, possibly due to contamination or sampling, while ALT had a false-negative case due to incomplete sampling. As previously observed, tumor grade is underestimated in cytologic specimens. Thus, ARX and ALT are the most promising markers to predict metastatic behavior in PanNETs, thereby warranting further validation in larger studies.
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http://dx.doi.org/10.1002/dc.24368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079001PMC
April 2020

Pancreatic cancer organoids recapitulate disease and allow personalized drug screening.

Proc Natl Acad Sci U S A 2019 Dec 9. Epub 2019 Dec 9.

Oncode Institute, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands;

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.
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http://dx.doi.org/10.1073/pnas.1911273116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936689PMC
December 2019

Tumor Seeding During Colonoscopy as a Possible Cause for Metachronous Colorectal Cancer.

Gastroenterology 2019 11 13;157(5):1222-1232.e4. Epub 2019 Aug 13.

Department of Gastroenterology and Hepatology, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address:

Background And Aims: In patients who have undergone surgery for colorectal cancer (CRC), 3% have recurrence of (metachronous) CRC. We investigated whether tumor seeding during colonoscopy (iatrogenic implantation of tumor cells in damaged mucosa) increases risk for metachronous CRC.

Methods: In a proof of principle study, we collected data from the Dutch National Pathology Registry for patients with a diagnosis of CRC from 2013 through 2015, with a second diagnosis of CRC within 6 months to 3.5 years after surgery. We reviewed pathology reports to identify likely metachronous CRC (histologically proven adenocarcinoma located elsewhere in the colon or rectum from the surgical anastomosis). For 22 patients fulfilling the inclusion criteria, we ascribed the most likely etiology to tumor seeding when endoscopic manipulations, such as biopsies or polypectomy, occurred at the location where the metachronous tumor was subsequently detected, after endoscopic manipulation of the primary tumor. We collected clinical data from patients and compared molecular profiles of the primary and metachronous colorectal tumors using next-generation sequencing. We then examined the source of seeded tumor. We tested whether tumor cells stay behind in the working channel of the endoscope after biopsies of colorectal tumors, and whether these cells maintain viability in organoid cultures.

Results: In total, tumor seeding was suspected as the most likely etiology of metachronous CRC in 5 patients. Tumor tissues were available from 3 patients. An identical molecular signature was observed in the primary and metachronous colorectal tumors from all 3 patients. In 5 control cases with a different etiology of metachronous CRC, the molecular signature of the primary and metachronous tumor were completely different. Based on review of 2147 patient records, we estimated the risk of tumor seeding during colonoscopy to be 0.3%-0.6%. We demonstrated that the working channel of the colonoscope becomes contaminated with viable tumor cells during biopsy collection. Subsequent instruments introduced through this working channel also became contaminated. These cells were shown to maintain their proliferative potential.

Conclusions: In an analysis of primary and secondary tumors from patients with metachronous CRC, we found that primary tumor cells might be seeded in a new location after biopsy of the primary tumor. Although our study does not eliminate other possibilities of transmission, our findings and experiments support the hypothesis that tumor seeding can occur during colonoscopy via the working channel of the endoscope. The possibility of iatrogenic seeding seems low. However, our findings compel awareness on this potentially preventable cause of metachronous CRC.
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http://dx.doi.org/10.1053/j.gastro.2019.07.062DOI Listing
November 2019

Enhancer signatures stratify and predict outcomes of non-functional pancreatic neuroendocrine tumors.

Nat Med 2019 08 1;25(8):1260-1265. Epub 2019 Jul 1.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Most pancreatic neuroendocrine tumors (PNETs) do not produce excess hormones and are therefore considered 'non-functional'. As clinical behaviors vary widely and distant metastases are eventually lethal, biological classifications might guide treatment. Using enhancer maps to infer gene regulatory programs, we find that non-functional PNETs fall into two major subtypes, with epigenomes and transcriptomes that partially resemble islet α- and β-cells. Transcription factors ARX and PDX1 specify these normal cells, respectively, and 84% of 142 non-functional PNETs expressed one or the other factor, occasionally both. Among 103 cases, distant relapses occurred almost exclusively in patients with ARXPDX1 tumors and, within this subtype, in cases with alternative lengthening of telomeres. These markedly different outcomes belied similar clinical presentations and histology and, in one cohort, occurred irrespective of MEN1 mutation. This robust molecular stratification provides insight into cell lineage correlates of non-functional PNETs, accurately predicts disease course and can inform postoperative clinical decisions.
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http://dx.doi.org/10.1038/s41591-019-0493-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919319PMC
August 2019

Well-differentiated Pancreatic Neuroendocrine Tumor in a Patient With Familial Atypical Multiple Mole Melanoma Syndrome (FAMMM).

Am J Surg Pathol 2019 09;43(9):1297-1302

Departments of Pathology.

Germline mutations in CDKN2A result in Familial Atypical Multiple Mole Melanoma Syndrome (FAMMM), which is associated with an increased risk for pancreatic ductal adenocarcinoma and melanoma. CDKN2A is somatically inactivated in multiple neoplasms, raising the possibility that, although the data are not conclusive, germline CDKN2A mutation may also impose an increased risk for other neoplasms. We present a patient with a CDKN2A germline mutation (p16-Leiden mutation) and mosaicism for neurofibromatosis type 2, who presented with a small asymptomatic pancreatic lesion, detected during endoscopic ultrasound screening of the pancreas. After resection, the lesion was found to be a well-differentiated pancreatic neuroendocrine tumor (PanNET). Molecular analysis of the tumor showed somatic loss of the second allele, supporting a causal relation of the PanNET to the underlying FAMMM syndrome. Recent data, showing the association between certain single-nucleotide polymorphisms in the CDKN2A gene and an increased incidence for PanNET, further support a role for germline CDKN2A alterations in PanNET risk. We conclude that PanNETs can be a phenotypic expression of FAMMM syndrome. This can have implications for screening and for the diagnosis of pancreatic neoplasms in carriers of germline CDKN2A mutations.
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http://dx.doi.org/10.1097/PAS.0000000000001314DOI Listing
September 2019

Pancreatic acinar cell carcinoma is associated with germline mutations: a case report and literature review.

Cancer Biol Ther 2019 19;20(7):949-955. Epub 2019 Apr 19.

a Department of Pathology , Radboud Institute for Molecular Life Sciences, Radboud university medical center , Nijmegen , The Netherlands.

Acinar cell carcinoma (ACC) is a rare pancreatic neoplasm with dismal prognosis. Insights into the molecular basis of ACC can pave the way for the application of more effective, personalized therapies and detection of patients with hereditary predisposition. Molecular analysis revealed a germline (and ) mutation in a patient with a rare pancreatic ACC with extensive intraductal growth. Somatic loss of the wild-type allele in the tumor indicated the causal relationship of ACC with the germline defect. A thorough literature review identified another nine ACCs associated with germline mutation and two ACCs associated with germline mutation, resulting in a prevalence of germline mutations in almost 7% of ACCs. Moreover, somatic alterations are reported in 16% of sporadic ACCs. Overall, about one fifth (22%) of all pancreatic ACCs exhibit BRCA1/2 deficiency. This study underscores the important role of mutations in pancreatic ACC. All ACC patients should undergo genetic testing for mutations to identify carriers of pathogenic variants. This will allow to select patients that can benefit from targeted therapies directed against -deficient tumors and is also crucial as a referral to genetic screening for the relatives of affected individuals carrying germline alterations. ACC: acinar cell carcinoma; HBOC: Hereditary Breast and Ovarian Cancer; LOH: loss of heterozygosity; PARP: poly (ADP-ribose) polymerase; PDAC: pancreatic ductal adenocarcinoma; PP: pancreatic panniculitis; SD: standard deviation; WES: whole-exome sequencing.
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http://dx.doi.org/10.1080/15384047.2019.1595274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606020PMC
July 2020

Pseudomyxoma Peritonei After a Total Pancreatectomy for Intraductal Papillary Mucinous Neoplasm With Colloid Carcinoma in Lynch Syndrome.

Pancreas 2019 01;48(1):135-138

Radiology, St Antonius Hospital, Nieuwegein, the Netherlands.

We report a case of pseudomyxoma peritonei (PMP) arising in a 62-year-old male patient with Lynch syndrome (LS). The patient's medical history included an adenocarcinoma of the colon for which a right hemicolectomy was performed and a pancreatectomy due to an intraductal papillary mucinous neoplasm (IPMN) with invasive colloid carcinoma. It was considered that the PMP could be a metastasis of the earlier colonic or pancreatic carcinoma. The pancreatic carcinoma, colon carcinoma, and PMP tissues were examined, and immunohistochemical and molecular analyses were performed to determine the PMP origin. Histopathologic examination revealed morphological similarities with the pancreatic colloid carcinoma, and further immunohistochemical and molecular analyses, including a shared GNAS mutation, confirmed the pancreatic origin of the PMP. In conclusion, this is a unique case of a patient with LS presenting with PMP originating from an IPMN with invasive colloid carcinoma, several years after pancreatectomy. The present case has important diagnostic implications. The IPMN should be considered as a rare extracolonic manifestation of LS, and pancreatic carcinoma origin should be considered in patients presenting with PMP. This case report highlights the added value of molecular diagnostics in daily pathology practice.
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http://dx.doi.org/10.1097/MPA.0000000000001201DOI Listing
January 2019

Longitudinal analysis of colon crypt stem cell dynamics in sulindac treated Familial Adenomatous Polyposis patients.

Sci Rep 2017 09 20;7(1):11972. Epub 2017 Sep 20.

Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

The non-steroidal anti-inflammatory drug sulindac decreases size and number of adenomas after 4-6 months of treatment for familial adenomatous polyposis (FAP) patients. However, the underlying mechanism remains unknown. As stem cells are thought to be the tumor precursor cells, visualizing their behavior is crucial for monitoring tumor progression. Increased tag diversity in inactive genes is indicative of a protracted clonal evolution and consequently, increased risk for tumor formation. Therefore, the effect of sulindac on stem cell dynamics was studied. Normal appearing single crypts were laser microdissected in placebo- and sulindac- treated FAP patient tissue after which the methylation patterns were visualized by Next Generation Sequencing. A significant difference in tag diversity over time was found in the sulindac group compared to the placebo group (*p = 0.018), indicative of a shortened clonal evolution treated sulindac. The rate of change in tag diversity over time was correlated with polyp number change over time. No significant difference over time was observed in the percent methylation when comparing placebo vs sulindac. In conclusion, daily sulindac administration in FAP patients significantly altered colorectal stem cell dynamics, which might explain the chemopreventive action of this drug indicating that tag diversity may be used as a predictive biomarker.
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http://dx.doi.org/10.1038/s41598-017-11865-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607292PMC
September 2017

Absence of mutation in juvenile polyposis syndrome.

J Clin Pathol 2017 07 19;70(7):640. Epub 2017 Apr 19.

Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

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http://dx.doi.org/10.1136/jclinpath-2017-204449DOI Listing
July 2017

Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.

Proc Natl Acad Sci U S A 2016 10 5;113(42):11859-11864. Epub 2016 Oct 5.

Hubrecht Institute for Developmental Biology and Stem Cell Research, Royal Netherlands Academy of Arts and Sciences (KNAW), 3584 CT Utrecht, The Netherlands; University Medical Centre Utrecht, 3584 CT Utrecht, The Netherlands;

Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1 knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1 KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1 KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.
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http://dx.doi.org/10.1073/pnas.1614057113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081651PMC
October 2016

Stem cell dynamics and pretumor progression in the intestinal tract.

J Gastroenterol 2016 Sep 23;51(9):841-52. Epub 2016 Apr 23.

Department of Pathology, University Medical Center, 3508 GA, Utrecht, The Netherlands.

Colorectal carcinogenesis is a process that follows a stepwise cascade that goes from the normal to an invisible pretumor stage ultimately leading to grossly visible tumor progression. During pretumor progression, an increasing accumulation of genetic alterations occurs, by definition without visible manifestations. It is generally thought that stem cells in the crypt base are responsible for this initiation of colorectal cancer progression because they are the origin of the differentiated epithelial cells that occupy the crypt. Furthermore, they are characterized by a long life span that enables them to acquire these cumulative mutations. Recent studies visualized the dynamics of stem cells both in vitro and in vivo. Translating this work into clinical applications will contribute to the evaluation of patients' predisposition for colorectal carcinogenesis and may help in the design of preventive measures for high-risk groups. In this review, we outline the progress made in the research into tracing stem cell dynamics. Further, we highlight the importance and potential clinical value of tracing stem cell dynamics in pretumor progression.
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http://dx.doi.org/10.1007/s00535-016-1211-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990616PMC
September 2016

Mice lacking functional CD95-ligand display reduced proliferation of the intestinal epithelium without gross homeostatic alterations.

Med Mol Morphol 2016 Jun 23;49(2):110-8. Epub 2015 Dec 23.

Department of Surgery, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.

Homeostasis of the continuously self-renewing intestinal tract involves cell proliferation, migration, differentiation along the crypt-villus-axis and shedding of cells into the gut lumen. CD95-ligand (FAS-ligand, CD95L) is a cytokine that is known for its capacity to induce apoptosis by binding its cognate receptor, CD95 (Fas). More recently, it was discovered that CD95L can also induce other cellular responses, such as proliferation, differentiation and cell migration. CD95L is highly expressed in Paneth cells of the small intestine which are in close contact with intestinal stem cells. This suggests a potential role for CD95L in controlling stem cell function and, possibly, intestinal homeostasis. We analyzed the intestines of mice deficient for functional CD95L (gld) for potential alterations in the diversity of stem-cell-lineages and parameters of intestinal homeostasis. Stem cell diversity was assessed by analyzing methylation patterns of the non-transcribed mMYOD gene. Proliferation was analyzed by BrdU labeling and differentiation was assessed by immunohistochemistry. Of all parameters analyzed, only epithelial cell proliferation was significantly reduced in the small intestines of gld-mice, but not in their colons which lack CD95L expression. We conclude that CD95L has a proliferation-stimulating role during normal turnover of the small intestine, but has a marginal effect on overall intestinal homeostasis.
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http://dx.doi.org/10.1007/s00795-015-0129-9DOI Listing
June 2016

Organoid models of human and mouse ductal pancreatic cancer.

Cell 2015 Jan 31;160(1-2):324-38. Epub 2014 Dec 31.

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; Lustgarten Foundation Pancreatic Cancer Research Laboratory, Cold Spring Harbor, NY 11724, USA.

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.
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http://dx.doi.org/10.1016/j.cell.2014.12.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334572PMC
January 2015

Epithelial-specific loss of PTEN results in colorectal juvenile polyp formation and invasive cancer.

Am J Pathol 2014 Jan 6;184(1):86-91. Epub 2013 Nov 6.

European Cancer Stem Cell Research Institute, Cardiff School of Biosciences, Cardiff University, Cardiff, United Kingdom. Electronic address:

Cowden syndrome (CS) is a rare autosomal dominant cancer-prone disorder caused by germ-line mutation of the phosphatase and tensin homolog mutated on chromosome 10 (PTEN) tumor-suppressor gene. Affected patients commonly develop juvenile polyps, and show an elevated risk of developing colorectal cancers. The etiology of these peculiar polyps remains unclear, although previous work has suggested somatic PTEN alterations in the stroma of juvenile polyps. After a long latency period, we find epithelial-specific PTEN deletion to cause formation of juvenile polyps in the colorectum without stromal PTEN loss. More important, we find that these lesions closely recapitulate all of the characteristic histopathological features of juvenile polyps seen in patients with CS, including stromal alterations and dysplastic transformation to colorectal carcinoma. The stromal alterations we identify after epithelial-specific PTEN loss suggest that PTEN may be involved in altered epithelial-mesenchymal cross talk, which, in turn, predisposes to colorectal neoplasia and polyposis. Our transgenic model is the first to recapitulate colorectal juvenile polyposis in patients with CS. We conclude that stromal PTEN loss is not a prerequisite for the formation of juvenile polyps, and that colorectal juvenile polyps in CS are bona fide neoplastic precursor lesions.
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http://dx.doi.org/10.1016/j.ajpath.2013.10.003DOI Listing
January 2014

Monoclonal origin of primary unilateral multifocal pleomorphic adenoma of the parotid gland.

Hum Pathol 2013 May 17;44(5):923-6. Epub 2013 Jan 17.

Department of Otorhinolaryngology and Head & Neck Surgery, University Medical Center Utrecht, The Netherlands.

Primary multiple pleomorphic adenomas in a unilateral parotid gland in previously untreated patients is a rare finding, and little is known about the etiology and pathogenesis. Here, a highly unusual case of a primary multifocal pleomorphic adenoma consisting of 15 individual nodules is presented. It is shown that all nodes are clonally related and thus share a common cell of origin excluding an independent multifocal pathogenesis. Most likely, multifocal pleomorphic adenoma represents parasitic nodules that have been detached from a main nodule, which may have been the result of undisclosed trauma.
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http://dx.doi.org/10.1016/j.humpath.2012.10.008DOI Listing
May 2013

Immunophenotyping invasive breast cancer: paving the road for molecular imaging.

BMC Cancer 2012 Jun 13;12:240. Epub 2012 Jun 13.

Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Mammographic population screening in The Netherlands has increased the number of breast cancer patients with small and non-palpable breast tumors. Nevertheless, mammography is not ultimately sensitive and specific for distinct subtypes. Molecular imaging with targeted tracers might increase specificity and sensitivity of detection. Because development of new tracers is labor-intensive and costly, we searched for the smallest panel of tumor membrane markers that would allow detection of the wide spectrum of invasive breast cancers.

Methods: Tissue microarrays containing 483 invasive breast cancers were stained by immunohistochemistry for a selected set of membrane proteins known to be expressed in breast cancer.

Results: The combination of highly tumor-specific markers glucose transporter 1 (GLUT1), epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), hepatocyte growth factor receptor (MET), and carbonic anhydrase 9 (CAIX) 'detected' 45.5% of tumors, especially basal/triple negative and HER2-driven ductal cancers. Addition of markers with a 2-fold tumor-to-normal ratio increased the detection rate to 98%. Including only markers with >3 fold tumor-to-normal ratio (CD44v6) resulted in an 80% detection rate. The detection rate of the panel containing both tumor-specific and less tumor-specific markers was not dependent on age, tumor grade, tumor size, or lymph node status.

Conclusions: In search of the minimal panel of targeted probes needed for the highest possible detection rate, we showed that 80% of all breast cancers express at least one of a panel of membrane markers (CD44v6, GLUT1, EGFR, HER2, and IGF1-R) that may therefore be suitable for molecular imaging strategies. This study thereby serves as a starting point for further development of a set of antibody-based optical tracers with a high breast cancer detection rate.
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http://dx.doi.org/10.1186/1471-2407-12-240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3430576PMC
June 2012

Multivariate analysis of immunohistochemical evaluation of protein expression in pancreatic ductal adenocarcinoma reveals prognostic significance for persistent Smad4 expression only.

Cell Oncol (Dordr) 2012 Apr 18;35(2):119-26. Epub 2012 Feb 18.

Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis with a 5-year survival rate of <5% and an average survival of only 6 months. Although advances have been made in understanding the pathogenesis of PDAC in the last decades, overall survival has not changed. Various clinicopathological and immunohistological variables have been associated with survival time but the exact role that these variables play in relation to survival is not clear.

Methods And Results: To examine how the variables affected survival independently, multivariate analysis was conducted in a study group of 78 pancreatic ductal adenocarcinomas. The analysis included clinicopathological parameters and protein expression examined by immunohistochemistry of p53, Smad4, Axl, ALDH, MSH2, MSH6, MLH1 and PMS2. Lymph node ratio <0.2 (p = 0.004), tumor free resection margins (p = 0.044) and Smad4 expression (p = 0.004) were the only independent prognostic variables in the multivariate analysis. Expression of the other proteins examined was not significantly related to survival.

Conclusions: Discrepancies with other studies in this regard are likely due to differences in quantification of immunohistochemical staining and the lack of multivariate analysis. It underscores the importance to standardize the methods used for the application of immunohistochemistry in prognostic studies.
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http://dx.doi.org/10.1007/s13402-012-0072-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306569PMC
April 2012

Pancreatic ductal adenocarcinoma in hereditary diffuse gastric cancer. A case report.

Hum Pathol 2012 Mar 10;43(3):457-61. Epub 2011 Oct 10.

Department of Pathology, University Medical Center Utrecht, 3584CX Utrecht, The Netherlands.

Hereditary diffuse gastric cancer is an autosomal dominant cancer syndrome characterized by highly penetrant diffuse gastric cancer. It is caused by germ line mutations in CDH1, encoding the cell-cell adhesion protein E-cadherin. Pancreatic ductal adenocarcinoma is one of the most dismal malignancies in humans. Although absent E-cadherin expression in pancreatic ductal adenocarcinoma is related to a higher tumor grade and a worse prognosis, there have been no reports of pancreatic ductal adenocarcinoma associated with hereditary diffuse gastric cancer. Here, we describe a patient with hereditary diffuse gastric cancer who was subsequently diagnosed with pancreatic ductal adenocarcinoma. To investigate if the previously identified CDH1 germ line mutation initiated pancreatic ductal adenocarcinoma development, we performed mutational and proteomic analyses. We conclude that the pancreatic ductal adenocarcinoma did not occur in the context of the germ line CDH1 mutation but rather appeared as a sporadic event. Immunohistochemistry ultimately proved to be the most valuable tool of investigation as persistent CDH1 staining in the pancreatic ductal adenocarcinoma unequivocally revealed E-cadherin expression.
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http://dx.doi.org/10.1016/j.humpath.2011.06.008DOI Listing
March 2012

Aberrant intestinal stem cell lineage dynamics in Peutz-Jeghers syndrome and familial adenomatous polyposis consistent with protracted clonal evolution in the crypt.

Gut 2012 Jun 22;61(6):839-46. Epub 2011 Sep 22.

Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

Objective: Genetic predisposition to cancer in Peutz-Jeghers syndrome (PJS) and the role of germline serine-threonine kinase (LKB1) mutations are poorly understood. The authors studied the effect of germline LKB1 mutations on intestinal stem cell dynamics in unaffected flat PJS mucosa. Recent research has documented that the intestinal crypt houses multiple equipotent stem cell lineages. Lineages continuously compete through random drifts, while somatically inherited methylation patterns record clonal diversity.

Design: To study the effect of germline LKB1 mutations on clonal expansion, the authors performed quantitative analyses of cardiac-specific homeobox methylation pattern diversity in crypts isolated from unaffected colonic mucosa obtained from archival PJS patient material. The authors compared methylation density and methylation pattern diversity in patients with PJS to those in patients with familial adenomatous polyposis and age-matched controls.

Results: The percentage of total methylation is comparable between groups, but the number of unique methylation patterns is significantly increased for patients with familial adenomatous polyposis and patients with PJS compared to control subjects.

Conclusions: Monoallelic LKB1 loss is not silent and provokes a protracted clonal evolution in the crypt. The increased methylation pattern diversity observed in unaffected PJS mucosa predicts that premalignant lesions will arise at an accelerated pace compared to the general population.
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http://dx.doi.org/10.1136/gutjnl-2011-300622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564664PMC
June 2012

Analysis of LKB1 mutations and other molecular alterations in pancreatic acinar cell carcinoma.

Mod Pathol 2011 Sep 13;24(9):1229-36. Epub 2011 May 13.

Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

Acinar cell carcinoma is a rare non-ductal neoplasm of the pancreas with poorly defined molecular genetic features. Recently, biallelic inactivation of LKB1 was described in an acinar cell carcinoma of a Peutz-Jeghers patient carrying a heterozygous germline LKB1 mutation, and inhibition of mTOR signaling resulted in partial remission of the tumor. To explore the potential of mTOR inhibitors in sporadic acinar cell carcinoma, the LKB1 gene was investigated in five sporadic acinar cell carcinomas by sequence analysis, methylation analysis and mRNA expression. In addition, microsatellite instability and methylation of a number of tumor suppressor genes were investigated and KRAS, TP53, CDKN1A, SMAD4 and CTNNB1 were studied by mutation analysis and immunohistochemistry. No mutations, deletions or promoter hypermethylation of LKB1 were found in any of the sporadic acinar cell carcinomas, and mRNA expression of LKB1 was not altered. Amplifications at chromosome 20q and 19p were found in 100 and 80% of the cases, respectively. In addition, hypermethylation of one or more tumor suppressor genes was found in 80% of cases. One case harbored a TP53 mutation, and expression of SMAD4 and CTNNB1 was altered in one case each. No KRAS mutations or microsatellite instability were found. To conclude, no evidence for a role for LKB1 in tumorigenesis of sporadic pancreatic acinar cell carcinoma was found. However, copy number variations and hypermethylation were found in a majority of cases. Molecular pathways involved in acinar cell carcinoma-tumorigenesis differ from those involved in ductal pancreatic neoplasms. Further studies are needed to increase our understanding of molecular pathogenesis of acinar cell carcinoma, which may eventually result in development of new therapeutic targets.
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http://dx.doi.org/10.1038/modpathol.2011.83DOI Listing
September 2011

Diversity counts. Visualizing pretumor progression in the gastrointestinal tract.

Am J Clin Pathol 2011 Jun;135(6):878-88

Dept. of Pathology, University Medical Center Utrecht, Utrecht, the Netherlands.

Tumor progression is critically dependent on the selection of genetic alterations. This clonal evolution can be traced to the stage preceding visible tumor formation called pretumor progression, in which genetic change occurs without visible change. Recently, the identification of intestinal stem cell markers in animal models has made visualization of stem cells possible in vivo. Translating this work to the clinical setting by visualizing stem cells in patient material may allow us to understand differences in patients' vulnerability to cancer development and target preventive measures to high-risk groups. In this review article, we examine some of the analytic methods currently used in research settings tracing stem cell dynamics.
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http://dx.doi.org/10.1309/AJCPP3I5HDYWMHJADOI Listing
June 2011