Search

Background & Aims Risk stratification for adverse events, such as metastasis to lymph nodes, is based only on histologic features of tumors. We aimed to compare adverse outcomes of pedunculated vs nonpedunculated T1 colorectal cancers (CRC). Methods We performed a retrospective study of 1656 patients diagnosed with T1CRC from 2000 through 2014 at 14 hospitals in The Netherlands. The median follow-up time of patients was 42.5 months (interquartile range, 18.5–77.5 mo). We evaluated the association between tumor morphology and the primary composite end point, adverse outcome, adjusted for clinical variables, histologic variables, resection margins, and treatment approach. Adverse outcome was defined as metastasis to lymph nodes, distant metastases, local recurrence, or residual tissue. Secondary end points were tumor metastasis, recurrence, and incomplete resection. Results Adverse outcome occurred in 67 of 723 patients (9.3%) with pedunculated T1CRCs vs 155 of 933 patients (16.6%) with nonpedunculated T1CRCs. Pedunculated morphology was independently associated with decreased risk of adverse outcome (adjusted odds ratio [OR], 0.59; 95% CI, 0.42–0.83; P = .003). Metastasis, incomplete resection, and recurrence were observed in 5.8%, 4.6%, and 3.9% of pedunculated T1CRCs vs 10.6%, 8.0%, and 6.6% of nonpedunculated T1CRCs, respectively. Pedunculated morphology was independently associated with a reduced risk of metastasis (adjusted OR, 0.62; 95% CI, 0.41–0.94; P = .03), incomplete resection (adjusted OR, 0.57; 95% CI, 0.36–0.91; P = .02), and recurrence (adjusted hazard ratio, 0.52; 95% CI, 0.32–0.85; P = .009). Metastasis, incomplete resection, and recurrence did not differ significantly between low-risk pedunculated vs nonpedunculated T1CRCs (0.8% vs 2.9%, P = .38; 1.5% vs 0%, P = .99; 1.5% vs 0%; P = .99). However, incomplete resection and recurrence were significantly lower for high-risk pedunculated vs nonpedunculated T1CRCs (6.5% vs 12.5%; P = .007; 4.4% vs 8.6%; P = .03). Conclusions In a retrospective study of patients with T1CRC, we found pedunculated morphology to be associated independently with a decreased risk of adverse outcome in a T1CRC population at high risk of adverse outcome. Incorporating morphologic features of tumors in risk assessment could help predict outcomes of patients with T1CRC and help identify the best candidates for surgery.

Background: The natural behavior of flat low-grade (LGD) and indefinite dysplasia (IND) in patients with inflammatory bowel disease (IBD) remains uncertain and seems to be dependent on the interpretation of the pathologist. We studied the progression rate of flat LGD and IND to advanced neoplasia (high-grade dysplasia [HGD] or colorectal cancer [CRC]) before and after histopathological review by a panel of gastrointestinal expert pathologists. Methods: A nationwide pathology database was used to identify IBD patients with dysplasia in six Dutch university medical centers between 1990 and 2006. Medical charts of patients with recorded flat LGD or IND were reviewed. Histological slides from three university medical centers were reviewed by a panel of three expert gastrointestinal pathologists. Results: We identified 113 flat LGD patients and 26 flat IND patients. Advanced neoplasia was found in 18 flat LGD patients (16%) after a median follow-up of 48 months, resulting in a 5-year progression rate of 12%. Five IND patients (19%) developed advanced neoplasia after a median follow-up of 24 months, resulting in a 5-year progression rate of 21%. Review of 1547 histological slides from 87 patients resulted in an increase of the 5-year progression rate of flat LGD to advanced neoplasia to 37%, whereas the progression rate of IND decreased to 5%. Conclusions: A diagnosis of flat LGD that is confirmed by a panel of expert gastrointestinal pathologists is associated with a substantial risk of progression to advanced neoplasia, while confirmed IND is associated with a low risk of progression. (Inflamm Bowel Dis 2011;17:1108-1116)

Pancreatic cancer has a dismal prognosis. We have studied this cancer through the eyes of Charles Darwin: with the focus on how these tumors arise and evolve. With molecular techniques and analyses from evolution theory, we have definitively shown how cysts in the pancreas can result in pancreatic cancer. We also describe that when these cystic precursor lesions start to invade into the surrounding tissue, this is associated with mutations in the SMAD4 and TGFBR2. In addition, we studied the three-dimensional morphology of this cancer through tissue clearing techniques. We found that pancreatic cancer has multiple mechanisms of invasion in the surrounding tissue and that the cancerous cells often grow next to veins. This explains why pancreatic cancer has such a bad prognosis and why these tumors metastasize in a very early stage to the liver: because the pancreatic veins drain the blood from the pancreas to the liver and they can take easily take cancer cells with them.

Undifferentiated carcinoma with osteoclast-like giant cells (UCOGC), a variant of pancreatic ductal adenocarcinoma (PDAC), has a striking genetic similarity to PDAC but a significantly improved overall survival. We hypothesize that this difference could be due to the immune response to the tumor, and as such, we investigated the expression of PD-1, PD-L1, and CD163 in a series of UCOGC. To this aim, 27 pancreatic UCOGCs (11 pure and 16 PDAC-associated), 5 extrapancreatic tumors with osteoclast-like giant cells and 10 pancreatic anaplastic carcinomas were immunostained using antibodies against PD-1, PD-L1, and CD163. In pancreatic UCOGCs, PD-L1 was expressed in neoplastic cells of 17 (63%) of 27 cases, more often in cases with an associated PDAC (P =.04). Expression of PD-L1 was associated with poor prognosis, confirmed by multivariate analysis: patients with PD-L1–positive UCOGCs had a risk of all-cause mortality that was 3 times higher than did patients with PD-L1–negative UCOGCs (hazard ratio, 3.397; 95% confidence interval, 1.023-18.375; P =.034). PD-L1 expression on tumor cells was also associated with aberrant P53 expression (P =.035). PD-1 was expressed on rare lymphocytes in 12 UCOGCs (44.4%), mainly located at the tumor periphery. CD163 was expressed on histiocytes, with a diffuse and strong staining pattern in all UCOGCs. Extrapancreatic tumors with osteoclast-like giant cells showed very similar staining patterns for the same proteins. Anaplastic carcinomas have some similarities to UCOGCs, but PD-L1 has no prognostic roles. Our results may have important implications for immunotherapeutic strategies in UCOGCs; these tumors may also represent a model for future therapeutic approaches against PDAC. © 2018 Elsevier Inc.

Ductal adenocarcinoma of the pancreas is a very aggressive disease with a high mortality rate. Pancreatic carcinoma is the fourth leading cause of cancer-related death in Western countries, despite the fact this cancer accounts for only about 3% of all malignant tumors. Most pancreatic cancers (approximately 80%) are diagnosed at a late, incurable stage. The poor prognosis and late presentation of pancreatic cancer patients underscore the importance of early detection, which is the sine qua non for the fight against pancreatic cancer. The possibility for early detection of pancreatic cancer may be realized by increasing our knowledge of the histology and molecular genetics of precursor lesions of pancreatic cancer in conjunction with the development of sensitive and specific screening tests (both invasive and non-invasive) to detect early pancreatic cancer. In this thesis we examined the potential use of MicroRNAs (miRNAs) as a biomarker for the early detection of pancreatic cancer and investigated several signaling pathways to explore their role in pancreatic tumorigenesis and the development of its precursor lesions. We found out that aberrant miRNA expression is an early event in the multistage progression of pancreatic cancer, and specifically miR-155 warrants further evaluation as a biomarker for pancreatic cancer in clinical samples. Furthermore, we looked at the Notch and Axl pathway in the setting of pancreatic cancer. Both pathways play a significant role in pancreatic carcinogenesis. Inhibition of the Notch or Axl pathway, in a panel of pancreatic cancer cell lines, resulted in significant reduction in cell viability, anchorage independent growth, as well as attenuation of migratory and invasive properties. These reports support and emphasize an important function of Notch and Axl in pancreatic cancer and both signaling pathways might be a promising therapeutic strategy in this carcinoma. Finally, we have characterized DNA Damage Response markers (DDR) in PanIN lesions to determine whether or not these are involved in early pancreatic neoplasia. Activation of the ATM-Chk2 checkpoint pathways was commonly observed in PanIN lesions, which suggest that ATM and Chk2 activation at early stage of pancreatic tumorigenesis suppresses tumor progression. Loss of p53 function appears to be a critical determinant for bypassing this checkpoint and the subsequent progression to invasive adenocarcinoma. In summary, the above studies in this thesis describe potential clinically useful biomarkers for pancreatic carcinogenesis. Moreover, our increased knowledge of the Notch and Axl signaling pathway in pancreatic tumorigenesis may provide the basis for developing more sensitive screening strategies and the identification of new drug targets enabling directed drug design. All this will enable us to develop better tools for primary and secondary prevention of pancreatic cancer, as well as improve existing tools for early diagnosis.

Gastric cancer is a result from the combination of environmental factors and an accumulation of specific genetic alterations, and affects mainly the older population. It is known that genetic factors play a more important role in early onset gastric cancers than in conventional gastric cancer patients as they have experienced less exposure to environmental factors. It is postulated that EOGC is a key tool in the explanation of the role of genetic changes in gastric carcinogenesis. The specific pathogenetic changes underlying gastric carcinogenesis remain largely unknown. The aim of this thesis is to uncover genetic alterations relevant to gastric carcinogenesis and to elucidate the role of the genes and proteins involved in the progress of cancer in different sub-types of gastric cancers. Additionally, we try to find the best approach for early diagnostics of gastric stump cancers. Gastric cancer is considered a multistep process and multifactor process. An overview of the involved factors as well as important genes in gastric carcinogenesis is provided in chapters 1 and 2. In these chapters the background of gastric cancer is extensively discussed. Additionally in these chapters the concept of early onset gastric cancer is introduced together with the reasoning behind the study of this subtype of gastric cancer. Chapter 3 focuses on a comparison between molecular alterations occurring in primary gastric cancers, corresponding xenografts and gastric carcinoma cell lines. Chapter 4 takes a step towards a better understanding of the progression of gastric carcinogenesis in early onset and conventional gastric cancers. Using Western blot, Q-PCR and sequencing analysis, we examined the correlation between two critical molecules in the development and progression of gastric cancer: COX-2 and E-cadherin. The relationship between these two molecules was examined in vitro in a panel of cell lines and in vivo using tissue microarrays. In chapter 5 we examine the COX-2 -765 G/C promoter polymorphism in different subtypes of gastric cancer. Real time PCR, sequencing analysis and immunohistochemical staining on tissue microarrays were used to find differences between EOGC, conventional gastric cancer and gastric stump cancer. Chapter 6 takes a look into the IL-1B -31 T/C polymorphism distribution in EOGC, conventional and gastric stump cancers using the same techniques as described in chapter 5. Chapter 7 reviews the literature on gastric stump cancer and its precursor lesions and focuses on the reliability of early diagnosis. Finally, chapters 8 and 9 provide brief summaries and a discussion of the thesis (in English and in Dutch respectively).

Hereditary breast cancer runs in families where several family members in different generations are affected. Most of these breast cancers are caused by mutations in the high penetrance genes BRCA1 and BRCA2 which account for about 5% of all breast cancers. However, mutations in BRCA1 and BRCA2 may be missed, and small families and sometimes insufficient medical records may cause uninformative family history. This thesis aimed to investigate the immunoprofile of hereditary breast cancers caused by germline mutations in the BRCA1 or BRCA2 genes in order to identify patients and families eligible for mutation screening. In addition, such studies on hereditary precursors could help to better understand hereditary breast carcinogenesis and identify new therapeutic targets. We started with evaluating a panel of clinicopathological variables to classify breast cancers as hereditary or sporadic using a multivariate approach. Most of the investigated markers were expressed in line with earlier data. However, we were the first to describe the high expression of EGFR in BRCA1 and BRCA2 related breast cancers. In addition, the observed high cyclin A expression is another useful proliferation marker in BRCA1 related breast cancers, and has been herewith established for the first time. Although our data were based on only five cases of BRCA2-related breast cancers, the cyclin D1 expression in these tumours was remarkably high. As such, most invasive breast carcinomas can be classified as sporadic or BRCA1 related with a high degree of certainty using a decision tree based on age, Ki67 and EGFR. Furthermore, we are the first to report that in BRCA1 and BRCA2 mutation carriers the immunoprofile of ductal carcinoma in situ (DCIS), a premalignant lesion, parallels the accompanying invasive cancer lesion, underlining that DCIS is probably a direct precursor lesion in these patients. In addition, crucial carcinogenetic events leading to these phenotypes apparently occur at an early stage in hereditary predisposed patients, most likely even before the stage of DCIS. We describe the novel finding of loss of expression of FANCD2, a Fanconi anaemia gene previously implicated in breast cancer in mice, in hereditary and sporadic invasive breast cancer, indicating that somatic inactivating (epi)genetic events in FANCD2 may occur in both sporadic and hereditary breast carcinogenesis. Our results do however not indicate that somatic (epi)genetic changes in FANCD2 are a frequent secondary carcinogenetic event in BRCA1 germline mutated patients. The expression of HIF-1?, the key regulator of the hypoxia response, in hereditary breast cancer is for the first time reported in comparison with sporadic breast cancer. 90% of BRCA1 germline mutation related breast cancers showed overexpression of HIF-1?, a percentage significantly higher than in sporadic controls. The overexpression of HIF-1? in BRCA1 related breast cancers is a frequent event and is likely hypoxia regulated, suggesting that the HIF-1? pathway plays a role in the BRCA-1 carcinogenesis and progression. All these findings will lead to improved discrimination of the BRCA1 and BRCA2 related breast cancer. Furthermore, the new biomarkers may serve as therapeutic targets for BRCA1 and BRCA2 related breast cancers.

Colorectal cancer (CRC) is an important cause death. In the Netherlands, approximately 10.000 patients are diagnosed with CRC each year. Rare hereditary gastrointestinal polyposis syndromes predisposing to CRC, including familial adenomatous polyposis (FAP), juvenile polyposis (JPS) and Peutz-Jeghers syndrome (PJS), have provided a model for the investigation of cancer initiation and progression in the general population. Many insights in the molecular genetic basis of CRC have emerged from studying these syndromes. The first part of this thesis is about duodenal polyposis in FAP and chemoprevention with NSAIDs. With improved management of colorectal disease and increased life expectancy of FAP patients, duodenal polyps and adenocarcinoma have become major health problems in these patients. Surveillance, endoscopic and surgical treatment and the value of NSAID chemoprevention for duodenal polyposis are reviewed. Results of NSAIDs on regression or prevention of duodenal adenomas in FAP appear disappointing. Expression of the main target of NSAIDs, cyclooxygenase-2 (COX-2) was studied and compared between colorectal and duodenal adenomas from patients with FAP. Levels of COX-2 appeared to be significantly higher in the duodenum than in the colorectum of FAP patients, which may explain the poorer response of duodenal adenomas to COX-2 inhibition, suggesting that higher dosages of COX-2 inhibitors are needed for treatment of duodenal adenomas. The second part of this thesis focuses on clinical and molecular aspects of JPS. We performed person-year analysis to define the magnitude of risk for gastrointestinal cancer in JPS. A relative risk of colorectal cancer of 34 and a cumulative life-time risk of 38.7% in JPS was found. JPS is caused by germline mutation of the SMAD4 or BMPR1A gene, but a germline mutation is found in only 30-40% of patients. Likely, alternative ways of inactivation of these genes exist. Therefore, we used MLPA to investigate whether germline deletion of these genes may cause JPS. By combining sequencing and MLPA, a germline defect in SMAD4 or BMPR1A was found in almost 50% of the patients. Nine mutations (33.3%) were detected by direct sequencing, whereas four germline hemizygous large genomic deletion (14.8%) were found using MLPA. Prevention of polyp and neoplasia development in juvenile polyposis using chemoprevention may be useful in the treatment of these patients. We measured expression of COX-2 in juvenile polyps to investigate the potential value of chemoprevention by COX-2 inhibition. Increased levels of COX-2 were found in juvenile polyps from JPS patients, suggesting that chemoprevention using COX-2 inhibition can be beneficial in JPS. Lastly, we studied molecular aspects of juvenile polyps from patients with a SMAD4 germline defect. Focal reduction or loss of SMAD4 expression was present in the epithelium of about half of the juvenile polyps from SMAD4 mutation carriers. LOH or somatic mutation of the wild type SMAD4 allele was found in cases with aberrant SMAD4 protein expression. These findings do not support a “landscaper” mechanism for SMAD4, although stromal haploinsufficiency or loss of SMAD4 may still have a role in polyp initiation and epithelial loss may only occur at a later stage in polyp progression. Surprisingly, loss of epithelial SMAD4 immunostaining did not correlate with dysplasia in polyps, suggesting that loss of the wild type allele is not obligatory for dysplastic change in juvenile polyps, not fitting with the typical tumor-suppressor/“gatekeeper” role for SMAD4, as previously suggested.

Diabetes mellitus is a severe and rapidly growing problem in health care, accounting for approximately 150 million patients worldwide. Patients with diabetes are at increased risk to develop diabetic nephropathy, which is currently the most important cause of end-stage renal disease in large parts of the world. The natural course of diabetic nephropathy is unpredictable and the pathogenesis of progression is not completely understood. Recently, connective tissue growth factor (CTGF) and bone morphogenetic proteins (BMPs) have emerged as key players in renal disease. The studies presented in this thesis aimed to elucidate the role of CTGF and BMPs, and their possible interaction in diabetic nephropathy. Both urinary CTGF excretion and plasma CTGF were increased in patients with diabetic nephropathy and were correlated with clinical markers of renal disease. The association of elevated CTGF levels with diabetic nephropathy proved to be at least as strong as that of the established risk factors hypertension and hyperglycemia. Next, in a prospective study, baseline plasma CTGF was identified as an independent predictor of end-stage renal disease and mortality in patients with diabetic nephropathy. Moreover, in diabetic patients with nephrotic range albuminuria, plasma CTGF was the only parameter predicting end-stage renal disease. These observations suggest that CTGF in urine and plasma might find clinical application as a biomarker. To study CTGF involvement in the pathogenesis of diabetic nephropathy, we investigated development of diabetic nephropathy in the context of high and low levels of CTGF. In transgenic CTGF+/- mice, which express 50% lower CTGF levels than wild-type mice, structural and functional changes of diabetic nephropathy were significantly attenuated. This was exemplified by less severe albuminuria, absence of glomerular basement membrane thickening, and preserved matrix metalloproteinase activity in diabetic CTGF+/- mice. Interestingly, we observed that CTGF inhibited the signaling activity and target gene expression of BMP-7, both in diabetic mice as well as in cultured renal cells. Thus, overexpression of CTGF plays a role in the pathogenesis of diabetic nephropathy, and this appears to involve inhibition of the antifibrotic activity of BMP-7. Another novel aspect in the pathogenesis of renal fibrosis where we identified involvement of BMPs is the contribution of circulating bone-marrow derived myofibroblast progenitor cells (MFPC). We showed that the number of MFPC is increased in patients with diabetes as compared to matched healthy control subjects, and that diabetes-derived MFPC were deficient in BMP-6 expression. We demonstrated that BMP-6, like BMP-7, could antagonize profibrotic activity of transforming growth factor-beta in renal cells. The direct impact of BMP-6 on MFPC and renal fibrosis was revealed in BMP-6 deficient mice, which developed more severe renal fibrosis after unilateral ureteral obstruction, in association with higher numbers of circulating MFPC. In conclusion, the studies in this thesis have identified CTGF and BMPs as determinants of renal fibrosis, and in particular diabetic nephropathy. We suggest that restoration of growth factor balance might constitute a novel strategy for treatment of diabetic nephropathy.

Gastrointestinal polyposis syndromes are characterized by the development of gastrointestinal polyps and an increased risk for neoplastic transformation. Peutz-Jeghers syndrome is a polyposis syndrome characterized by the development of gastrointestinal hamartomas; the molecular and histopathological origin of these polyps is unclear and hence the pre-malignant potential, if any, of these polyps remains unresolved. Current follow-up protocols mandate removal of polyps to decrease risk of intussusception as well as to decrease risk of neoplastic transformation. The current thesis investigates the premalignant potential of these polyps and proposes that in fact the polyps are an epiphenomenon to the malignant condition. Polyp removal therefore does not necessarily reduce cancer risk. Furthermore we show that abnormalities can be demonstrated in normal (id est unaffected) epithelia in Peutz-Jeghers syndrome that may suggest protracted clonal evolution at the level of the intestinal stem cell niche. Altered clonal evolution in the crypt would jointly explain the development of the benign gastrointestinal hamartomas as well as an increased risk for neoplastic transformation in these patients. Lastly, we show that the linchpin component of the Wnt signalling pathway, beta-catenin, appears on the plasma membrane upon Wnt treatment in Wnt-responsive cells. This suggests a previously unappreciated step in a signalling pathway frequently deregulated in familial and sporadic forms of colorectal cancer.