Digital image analysis of HER2 immunohistochemistry in gastric- and oesophageal adenocarcinoma: a validation study on biopsies and surgical specimens.

AIMS: To test the validity of diagnostics incorporating digital image analysis (DIA) for human epidermal growth factor 2 (HER2) immunohistochemistry (IHC) in gastro-oesophageal adenocarcinomas, as an alternative to current standard diagnostics using manual scoring. METHODS AND RESULTS: We included 319 consecutive gastro-oesophageal adenocarcinomas (232 biopsies and 87 surgical specimens). DIA was applied to determine HER2 IHC classification, using both standard breast cancer (BC) and modified gastro-oesophageal cancer (GEC) cut-offs. Consensus manual scores were established by four independent observers. Chromogenic in-situ hybridization (CISH) was performed on all 2+ cases by manual scoring, DIA or both. HER2 status was considered positive in 3+ and CISH-positive 2+ cases. Overall agreement between DIA and consensus manual scores was 76.5% (weighted κ = 0.66, BC cut-offs) and 85.6% (weighted κ = 0.80, GEC cut-offs). Agreement was similar for biopsies and surgical specimens. All disagreement occurred in the manual IHC equivocal cases. DIA resulted in a reduction of 2+ cases: 75.8% with BC cut-offs and 46.5% with GEC cut-offs. HER2 status was positive in 48 cases (15%) with standard diagnostics and DIA using GEC cut-offs, and 46 cases (14.4%) using BC cut-offs (all with CISH in 2+ cases). Considering standard diagnostics as a reference, DIA showed 93.8% sensitivity and 99.6% specificity (BC cut-offs) or 97.9% sensitivity and 99.6% specificity (GEC cut-offs). CONCLUSIONS: DIA is a reliable and feasible alternative to manual HER2 IHC scoring in gastro-oesophageal adenocarcinoma, both in biopsies and surgical specimens, leading to a reduction of 2+ cases for which subsequent ISH testing is required.

Clinical consequences of diagnostic variability in the histopathological evaluation of early rectal cancer.

INTRODUCTION: In early rectal cancer, organ sparing treatment strategies such as local excision have gained popularity. The necessity of radical surgery is based on the histopathological evaluation of the local excision specimen. This study aimed to describe diagnostic variability between pathologists, and its impact on treatment allocation in patients with locally excised early rectal cancer. MATERIALS AND METHODS: Patients with locally excised pT1-2 rectal cancer were included in this prospective cohort study. Both quantitative measures and histopathological risk factors (i.e. poor differentiation, deep submucosal invasion, and lymphatic- or venous invasion) were evaluated. Interobserver variability was reported by both percentages and Fleiss' Kappa- (ĸ) or intra-class correlation coefficients. RESULTS: A total of 126 patients were included. Ninety-four percent of the original histopathological reports contained all required parameters. In 73 of the 126 (57.9%) patients, at least one discordant parameter was observed, which regarded histopathological risk factors for lymph node metastases in 36 patients (28.6%). Interobserver agreement among different variables varied between 74% and 95% or ĸ 0.530-0.962. The assessment of lymphovascular invasion showed discordances in 26% (ĸ = 0.530, 95% CI 0.375-0.684) of the cases. In fourteen (11%) patients, discordances led to a change in treatment strategy. CONCLUSION: This study demonstrated that there is substantial interobserver variability between pathologists, especially in the assessment of lymphovascular invasion. Pathologists play a key role in treatment allocation after local excision of early rectal cancer, therefore interobserver variability needs to be reduced to decrease the number of patients that are over- or undertreated.

Sex Differences in Neoplastic Progression in Barrett's Esophagus: A Multicenter Prospective Cohort Study.

Recommendations in Barrett's esophagus (BE) guidelines are mainly based on male patients. We aimed to evaluate sex differences in BE patients in (1) probability of and (2) time to neoplastic progression, and (3) differences in the stage distribution of neoplasia. We conducted a multicenter prospective cohort study including 868 BE patients. Cox regression modeling and accelerated failure time modeling were used to estimate the sex differences. Neoplastic progression was defined as high-grade dysplasia (HGD) and/or esophageal adenocarcinoma (EAC). Among the 639 (74%) males and 229 females that were included (median follow-up 7.1 years), 61 (7.0%) developed HGD/EAC. Neoplastic progression risk was estimated to be twice as high among males (HR 2.26, 95% CI 1.11-4.62) than females. The risk of HGD was found to be higher in males (HR 3.76, 95% CI 1.33-10.6). Time to HGD/EAC (AR 0.52, 95% CI 0.29-0.95) and HGD (AR 0.40, 95% CI 0.19-0.86) was shorter in males. Females had proportionally more EAC than HGD and tended to have higher stages of neoplasia at diagnosis. In conclusion, both the risk of and time to neoplastic progression were higher in males. However, females were proportionally more often diagnosed with (advanced) EAC. We should strive for improved neoplastic risk stratification per individual BE patient, incorporating sex disparities into new prediction models.

Pathologic diagnostics of HER2 positivity in gastroesophageal adenocarcinoma.

OBJECTIVES: The human epidermal growth factor receptor 2 (HER2) oncogene shows overexpression in 15% to 30% of gastroesophageal adenocarcinomas. Targeted anti-HER2 therapy with trastuzumab has been recently validated in advanced gastric and gastroesophageal junction cancer treatment. A standardized modified scoring system was recently introduced for gastroesophageal HER2 scoring. We aimed to validate this scoring system, including an analysis of interobserver variability of immunohistochemistry (IHC) scoring. METHODS: In total, 323 patients with histologically confirmed invasive gastric or esophageal adenocarcinoma were examined for HER2 by IHC and chromogenic in situ hybridization (CISH). IHC 3 + or IHC 2 +/CISH positive tumors were considered HER2 positive. Interobserver variability on IHC scoring using the currently standard modified HER2 scoring system was determined among three clinical pathologists. Clinicopathologic characteristics were retrospectively retrieved from the patient records. RESULTS: HER2 positivity was found in 50 (15.5%) of 323 patients. Interobserver agreement on IHC scoring was high (κ = 0.78). Most disagreement was found in diffuse or mixed tumor types and in weak to moderate stained samples (IHC 2 +). The HER2 IHC scoring system is sensitive in differentiating HER2 status before ISH. CONCLUSIONS: The currently used standardized HER2 scoring system is an excellent, clinically applicable method to establish HER2 status in appropriately educated and trained pathologists.

Cure of Barrett's carcinoma by ifosfamide chemotherapy.

The prognosis of patients with oesophageal cancer is poor, with an overall 5-year survival rate below 15%. The best chance for cure of patients with oesophageal cancer is surgical resection. However, more than 50% of patients have inoperable disease and can only be palliated for dysphagia. Some of these patients participate in studies investigating the activity of single-agent or combination chemotherapy. We report a patient who was cured of metastatic adenocarcinoma in Barrett's oesophagus by six courses of ifosfamide, a chemotherapeutic agent with little or no activity in other patients with adenocarcinoma of the oesophagus or gastro-oesophageal junction. After a follow-up of 13 years and 7 months, no evidence of tumour recurrence was found, while biopsies from the Barrett's oesophagus revealed only low-grade dysplasia. This case obviously raises the question as to how patients with inoperable oesophageal carcinoma can sometimes be cured by chemotherapy alone.

Limited diagnostic value of microsatellite instability associated pathology features in colorectal cancer.

To determine the diagnostic test characteristics and inter-observer variation of pathology features for identifying high microsatellite instability (MSI-H) colorectal cancer (CRC). Six pathologists blindly evaluated 177 CRC for the presence of MSI-H associated pathology features. Inter-observer agreement was determined by using Kappa-statistics. In the first random 88/177 cases, mucinous carcinoma, tumor-infiltrating lymphocytes (TIL) and Crohns-like infiltrate (CLI) were the best discriminators between MSI-H and microsatellite stable CRC [OR 5.6 (95 % CI 1.7-19), 5.4 (1.8-17) and 3.5 (1.1-11), respectively], with high specificity (89-91 %). The sensitivities for MSI-H, however, were low (31-41 %). In addition, inter-observer agreement was moderate for TIL and CLI (κ 0.38 and 0.48, respectively), but very good for mucinous carcinoma (κ 0.86). Interpretation of overall histopathology as suggestive for MSI-H performed better than any individual feature; OR 15 (5.2-44), and area under the curve 0.79. However, inter-observer agreement was moderate (κ 0.53). In the second set, TIL and CLI were scored according to updated scoring systems. Although both remained the best individual discriminators, test characteristics and inter-observer agreement did not improve. MSI-H pathology features have moderate accuracy for identifying MSI-H CRC, and are identified with moderate inter-observer agreement. These findings highlight the limitations of clinical strategies, such as the revised Bethesda guidelines, which incorporate the MSI-H associated pathology features in their strategy to identify persons with lynch syndrome.

Genomic analysis of Barrett's esophagus after ablative therapy: persistence of genetic alterations at tumor suppressor loci.

Barrett's esophagus (BE) is a major predisposing factor for the development of esophageal adenocarcinoma. Current strategies for treatment of BE, both dysplastic and nondysplastic, include photodynamic therapy (PDT) and argon plasma coagulation (APC). However, the effect of ablative therapy at the genetic level is unclear. We performed loss of heterozygosity (LOH) analysis of BE in baseline and follow-up biopsy specimens from 21 patients with BE (17 male, 4 female) treated with PDT and/or APC. At baseline, 14 patients had intestinal metaplasia without dysplasia (MET), 4 low-grade dysplasia (LGD) and 3 high-grade dysplasia (HGD). LOH was assessed using a panel of 9 polymorphic markers for evaluation of the P53 gene on 17p, P16 on 9p, DCC and SMAD4 on 18q and the APC gene on 5q. The tissue specimens obtained at baseline (t = 0) were analysed, as well as the first (t = 1; mean interval: 4 months) and last (t = 2; mean interval: 8 months) available biopsy with residual or recurrent BE after ablation. At t = 0, allelic loss was detected of 5q in 27%, 9p in 56%, 17p in 31% and 18q in 6% of informative cases. At t = 1 (18 patients with persistent MET and 3 with LGD) and at t = 2 (8 MET, 2 LGD), the LOH patterns were not statistically different from t = 0. Further, multiple genetic lineages before and after therapy were detected in 15 cases illustrating the multiclonal nature of BE. We conclude that recurrent and/or persistent BE after ablative therapy still contains genetic alterations associated with malignant progression to cancer. Therefore, the goal of treatment should be the complete elimination of Barrett's mucosa.

Molecular evaluation of ablative therapy of Barrett's oesophagus.

Barrett's oesophagus is a major risk factor for developing oesophageal adenocarcinoma. Ablation by argon plasma coagulation (APC) and photodynamic therapy (PDT) is currently under investigation for the removal of metaplastic and dysplastic Barrett's oesophagus. This study examined the effect of ablative therapy on Barrett's oesophagus at cell-cycle and genetic levels. The premalignant potential of residual or recurring Barrett's oesophagus was assessed by p53 immunohistochemistry, Ki67-related proliferative capacity, and DNA ploidy status (ie an abnormal chromosome 1 number) as measured by interphase in situ hybridization. Twenty-nine patients with Barrett's oesophagus (23 male and 6 female, mean age 58 years, mean length of Barrett's oesophagus 4 cm) were treated with APC or PDT. Intestinal metaplasia without dysplasia was present in 16 patients, low-grade dysplasia in five, and high-grade dysplasia in eight patients. Biopsy samples were obtained at regular intervals (mean follow-up 20 months, range 6-36 months). One month after the first ablation, Barrett's oesophagus was no longer identified, either endoscopically or histologically, in nine patients (32%). At this time point, significant down-grading was achieved for abnormal chromosome 1 numbers (p = 0.020) and Ki67-defined proliferation (p = 0.002). Patients with residual Barrett's oesophagus were additionally treated with APC, resulting in the elimination of Barrett's oesophagus in 76% of all patients. However, at the last follow-up endoscopy, metaplasia without dysplasia was still present in five patients, and low- and high-grade dysplasia were each present in one patient. An abnormal chromosome 1 number and p53 protein overexpression were detected only in the high-grade dysplastic lesion, but increased proliferation was still present in the majority of these persisting cases. Although endoscopic removal of Barrett's oesophagus by ablative therapies is possible in the majority of patients, histologically complete elimination cannot be achieved in all cases. Persistent Barrett's oesophagus may still harbour molecular aberrations and must therefore be considered still to be at risk of progression to adenocarcinoma.