Marjon Kerkhof

Aneuploidy and overexpression of Ki67 and p53 as markers for neoplastic progression in Barrett's esophagus: A case-control study

OBJECTIVES:Surveillance of patients with Barretts esophagus (BE) aims at early detection and treatment of neoplastic changes, particularly esophageal adenocarcinoma (EAC). The histological evaluation of biopsy samples has its limitations, and biomarkers may improve early identification of BE patients at risk for progression to EAC. The aim of this study was to determine the predictive value of p53, Ki67, and aneuploidy as markers of neoplastic progression in BE.METHODS:A total of 27 BE patients with histologically proven progression to high-grade dysplasia (HGD) or EAC (cases) and 27 BE patients without progression (controls) were selected and matched for age, gender, and duration of follow-up. Dysplasia grade was determined in 212 biopsy samples obtained during surveillance endoscopies from cases and in 231 biopsy samples collected from controls. DNA ploidy status was determined by flow cytometry, whereas Ki67 and p53 expression was determined by immunohistochemistry. Hazard ratios (HRs) were calculated by Cox regression adjusted for potentially confounding variables.RESULTS:A univariate analysis showed that low-grade dysplasia (LGD) increased the risk of developing HGD/EAC compared with no dysplasia (HR 3.6; 95% confidence interval (CI): 1.6 – 8.1). Aneuploidy (HR 3.5; 95% CI: 1.3–9.4), strong Ki67 overexpression (HR 5.2; 95% CI: 1.5–17.6), and moderate p53 overexpression (HR 6.5; 95% CI: 2.5–17.1) were also associated with an increased risk of developing HGD/EAC, independent of the histological result. A multivariable analysis showed that in the presence of LGD, p53 overexpression, and to a lesser extent, Ki67 overexpression remained important risk factors for neoplastic progression, whereas aneuploidy was no longer predictive.CONCLUSIONS:p53 overexpression and, to a lesser extent, Ki67 overexpression could predict neoplastic progression in BE irrespective of the histological result. These markers may be useful for identifying patients at an increased risk of developing EAC, either alone or used as a panel.

Risk Factors for the Development of Esophageal Adenocarcinoma in Barrett's Esophagus

OBJECTIVE:To identify risk factors for esophageal adenocarcinoma (EAC) in patients with Barretts esophagus (BE).METHODS:A hospital-based case-control study was performed in which 91 cases with EAC and 244 controls with histologically confirmed BE (>2 cm) with no dysplasia or low-grade dysplasia were included. Information on demographic, anthropometric, and lifestyle characteristics, physical activity levels, working posture, family history, gastroesophageal reflux disease (GERD) symptoms, and medication use was collected by questionnaire.RESULTS:Cases more often were current smokers (odds ratio 3.7, 95% confidence interval 1.4–9.9), more often had a body mass index >25 assessed at age 20 (2.6, 1.2–5.5), and more frequently had been working in a stooped posture at age 20 (2.0, 1.1–3.9), compared to controls. In addition, cases less often experienced symptoms of heartburn (0.3, 0.2–0.5) and less frequently used proton pump inhibitors (0.1, 0.05–0.2), compared to controls, whereas use of nonsteroidal anti-inflammatory drugs/aspirin was more common among cases (1.8, 1.1–3.2). Cases more often were men, compared to controls (91% vs 67%, p < 0.001).CONCLUSION:In patients with BE, the risk of EAC is related to risk factors for GERD, which is, however, asymptomatic. As these risk factors are common in Western countries, they are probably not helpful in individualization of surveillance intervals.

Aneuploidy and high expression of p53 and Ki67 is associated with neoplastic progression in Barrett esophagus

Unless detected at an early stage, esophageal adenocarcinoma (EAC) has a poor prognosis. Changes in cellular DNA content and expression levels of p53 and Ki67 in Barrett esophagus (BE) are associated with the development EAC and might serve as markers to identify EAC at an early stage. The aim of this study was to examine the presence of these three markers in various steps of neoplastic progression in BE towards EAC. Dysplasia was graded in 212 biopsy sets taken during follow-up upper endoscopy in 27 patients in whom ultimately high-grade dysplasia (HGD) or EAC was detected. Ploidy status was determined by flow cytometry, whereas Ki67 and p53 expression were determined by immunohistochemistry. Smoothing splines were used to analyze trends in time. We found an increasing fraction of Ki67 overexpression and, to a lesser extent, abnormal DNA content in biopsies closer to the time-point of detecting HGD/EAC in BE, suggesting the potential value of these biomarkers in identifying patients at increased risk of progression towards HGD/EAC. Accumulation of p53 was seen several years before development of HGD/EAC, and may therefore be an early marker in BE at a stage when dysplasia is not yet detected. A prospective follow-up study is needed to confirm these findings.

Biomarkers for risk stratification of neoplastic progression in Barrett esophagus

Barrett esophagus (BE) is caused by chronic gastroesophageal reflux and predisposes to the development of esophageal adenocarcinoma through different grades of dysplasia. Only a subset of BE patients will finally develop esophageal adenocarcinoma. The majority will therefore not benefit from an endoscopic surveillance program, based on the histological identification of dysplasia. Several studies have been performed to find additional biomarkers that can be used to detect the subgroup of patients with an increased risk of developing malignancy in BE. In this review, we will summarize the most promising tissue biomarkers, i.e. proliferation/cell cycle proteins, tumor suppressor genes, adhesion molecules, DNA ploidy status and inflammation associated markers, that can be used for risk stratification in BE, and discuss their respective clinical application.

Never forget aceruloplasminemia in case of highly suggestive Wilson's disease score.

A 49-year-old male presented with a 1-year history of combined bulbar and pseudobulbar dysarthria, involuntary movements, and gait instability. Laboratory evaluation showed a decreased serum ceruloplasmin of <0.06 g/L. Despite mild elevation of aspartate aminotransferase (54 E/L) and alanine aminotransferase (101 E/L), liver function (international normalized ratio, albumin, and bilirubin) was preserved. Magnetic resonance imaging (MRI) showed an abnormal attenuation of bilateral basal ganglia and cerebellar atrophy (Fig. 1). A brain computed tomography scan revealed hyperdensity of the basal ganglia and dentate nucleus. Because these findings were strongly suggestive of Wilson’s disease (WD), our workup continued to focus on further establishing this diagnosis. The ophthalmologist did not see Kayser-Fleischer (KF) rings, and 24-hour urine collection demonstrated a 0.6-umol copper loss per day (normal, 0.0-1.2 umol/day). According to the WD scoring system in the European Association for the Study of the Liver (EASL) guidelines, the diagnosis of WD was established (Table 1). To confirm this diagnosis and evaluate the degree of fibrosis, a liver biopsy was performed. However, copper staining on the liver biopsy specimen was negative. Determination of dry weight of copper was not available. Minor fibrosis and mild steatosis was noted without inflammation. In addition, extensive deposition of iron was noted, inconsistent with the diagnosis of WD (Fig. 2). Serum ferritin was elevated (2,908 ug/L) with a normal transferrin. The elevated ferritin in combination with the neurological symptoms, liver biopsy, MRI findings, and low ceruloplasmin was consistent with the diagnosis of aceruloplasminemia and less so with WD. At this point, a positive family history of aceruloplasminemia in the children of the patient’s maternal aunt was revealed. Finally, analysis for the ATP7B gene revealed no mutation and therefore did not support the diagnosis of WD. Aceruloplasminemia is an extremely rare (1:2,000,000) autosomal recessive disorder associated with low serum ceruloplasmin and neurological symptoms. In WD, neurological symptoms develop as a result of copper accumulation and in aceruloplasminemia as a result of iron accumulation in the central nervous system. In aceruloplasminemia, iron accumulation in brain and liver is the result of disturbances of iron metabolism because of loss-of-function mutations of the ceruloplasmin gene. These adults present with basal gangliar neurodegeneration (leading to dementia, dysarthria, and dystonia), retinal degeneration, diabetes mellitus, near-absent circulating serum ceruloplasmin, and elevated serum ferritin. Liver biopsy reveals normal hepatic architecture with abundant iron deposition without copper accumulation. In WD, copper accumulation in brain and liver is the result of defective biliary excretion of copper. Key features are liver disease, neuropsychiatric disturbances, and KF rings of the cornea. Dry weight of >250 lg/g of copper in a liver biopsy establishes the diagnosis, but normal values can be found because of inhomogeneous distribution of copper in the liver. Because clinical symptoms vary and no single test is specific, a WD scoring system based on all available tests was developed, with a good diagnostic accuracy (Table 1). According to the EASL guideline, a score of 4 points establishes the diagnosis of WD. This differs from the original scoring system, which defines this score as “highly likely” for the diagnosis of WD, thus forcing the clinician to consider an Abbreviations: EASL, European Association for the Study of the Liver; KF, Kayser-Fleischer; MRI, magnetic resonance imaging; WD, Wilson’s disease. From the Department of Gastroenterology and Hepatology, Albert Schweitzer hospital, Dordrecht, the Netherlands. Received April 12, 2013; accepted August 25, 2013. Address reprint requests to: Pieter Honkoop, M.D., Ph.D., Department of Gastroenterology and Hepatology, Albert Schweitzer Hospital, P.O. Box 444, 3300AK Dordrecht, the Netherlands. E-mail: p.honkoop@asz.nl; fax: 131 (0)78 6541544. Copyright VC 2014 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.26719 Potential conflict of interest: Nothing to report.

Diagnosis of T1 colorectal cancer in pedunculated polyps in daily clinical practice : a multicenter study

T1 colorectal cancer can be mimicked by pseudo-invasion in pedunculated polyps. British guidelines are currently one of the few which recommend diagnostic confirmation of T1 colorectal cancer by a second pathologist. The aim of this study was to provide insights into the accuracy of histological diagnosis of pedunculated T1 colorectal cancer in daily clinical practice. A sample of 128 cases diagnosed as pedunculated T1 colorectal cancer between 2000 and 2014 from 10 Dutch hospitals was selected for histological review. Firstly, two Dutch expert gastrointestinal pathologists reviewed all hematoxylin-eosin stained slides. In 20 cases the diagnosis T1 colorectal cancer was not confirmed (20/128; 16%). The discordant cases were subsequently discussed with a third Dutch gastrointestinal pathologist and a consensus diagnosis was agreed. The revised diagnoses were pseudo-invasion in 10 cases (10/128; 8%), high-grade dysplasia in 4 cases (4/128; 3%), and equivocal in 6 cases (6/128; 5%). To further validate the consensus diagnosis, the discordant cases were reviewed by an independent expert pathologist from the United Kingdom. A total of 39 cases were reviewed blindly including the 20 cases with a revised diagnosis and 19 control cases where the Dutch expert panel agreed with the original reporting pathologists diagnosis. In 19 of the 20 cases with a revised diagnosis the British pathologist agreed that T1 colorectal cancer could not be confirmed. Additionally, amongst the 19 control cases the British pathologist was unable to confirm T1 colorectal cancer in a further 4 cases and was equivocal in 3 cases. In conclusion, both generalist and expert pathologists experience diagnostic difficulty distinguishing pseudo-invasion and high-grade dysplasia from T1 colorectal cancer. In order to prevent overtreatment, review of the histology of pedunculated T1 colorectal cancers by a second pathologist should be considered with discussion of these cases at a multidisciplinary meeting.

Colorectal endoscopic full-thickness resection using a novel, flat-base over-the-scope clip: a prospective study

Background and study aims We aimed to evaluate the feasibility and safety of a new, flat-based over-the-scope clip (Padlock Clip) for colorectal endoscopic full-thickness resection (eFTR). Patients and methods We prospectively included 26 patients with lesions < 20 mm. Indications for eFTR were re-resection of the scar of a low risk malignant polyp (n = 11), recurrent adenoma in a non-lifting scar (n = 10), non-lifting polyp (n = 4), and an adenoma located in a diverticulum (n = 1). Results Technical success rate and full-thickness resection rate were 100 % (26/26) and 92 % (24/26), respectively. Median procedure time was 43 minutes (IQR 27 - 56). No complications occurred during the procedure; 3 complications (12 %) occurred within 48 hours, of which one was a perforation requiring laparoscopic suturing. Specimen volumes from eFTR of scar tissue where the original polyp had been ≥ 20 mm (n = 13) were smaller compared with those from non-scar resections or scars where the original polyps had been < 20 mm (n = 13) (median 0.8 vs. 1.5 cm3, P = 0.03). Conclusions In this first series of colorectal eFTR using the Padlock Clip, feasibility was demonstrated. It was relatively safe in view of surgery as the alternative treatment, but could still benefit from technical refinement. Future studies should explore for which indication this technique is most suitable. TRIAL REGISTRATION NTR5562 (Dutch Trial Register).

Pedunculated Morphology of T1 Colorectal Tumors Associates With Reduced Risk of Adverse Outcome

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.