Josiane C. Wink

Immunohistochemical Evaluation of a Panel of Tumor Cell Markers During Malignant Progression in Barrett Esophagus

Histopathologic grading of dysplasia in Barrett esophagus (BE) shows substantial interobserver and intraobserver variation. We used immunohistochemical analysis with a set of tumor cell markers, ie, epidermal growth factor receptor (EGFR), ERBB2 (HER2/neu), MYC, CDKN2A (p16), SMAD4, MET, CCND1 (cyclin D1), CTNNB1 (beta-catenin), and TP53 (p53), in histologic sections of endoscopic biopsies of 86 patients with BE in various stages of neoplastic progression. The markers, except SMAD4, were scored as 0 (<1% of cells stained), 1 (1%-25%), 2 (26%-50%), or 3 (>50%). All markers, except EGFR, showed a significant trend for immunohistochemical protein overexpression during malignant progression in BE (P <.01). When the successive stages along the metaplasia-low-grade dysplasia (LGD)-high-grade dysplasia (HGD)-adenocarcinoma axis were compared, protein overexpression of beta-catenin separated LGD from metaplasia, whereas protein overexpression of cyclin D1 and p53 discriminated HGD from LGD (all P <.001). beta-Catenin can be helpful for a diagnosis of LGD in BE, although it stains positively in a subset only, whereas p53 remains an appropriate marker to define HGD. In case of doubt, cyclin D1 can be added to separate LGD from HGD in BE.

Chromosomal and Microsatellite Instability of Adenocarcinomas and Dysplastic Lesions (dalm) in Ulcerative Colitis

Longstanding ulcerative colitis (UC) is associated with a high risk of developing UC-related colonic adenocarcinoma (UCC). These carcinomas originate from nonadenomatous dysplastic regions referred to as dysplasia associated lesion or mass (DALM). We evaluated chromosomal and microsatellite instability (MSI) in 21 DALM/UCCs. Chromosomal instability was determined by high-resolution array comparative genomic hybridization with a 3500-element BAC-PAC array. MSI was assessed with markers BAT25 and BAT26 and by immunohistochemical analysis of mismatch repair genes. Comparative genomic hybridization revealed frequent losses of array clones (>20% of tumors) at chromosome arms 4p, 5q, and 18q, frequent gains of array clones (>20% of tumors) were found at 1q, 5p, 6p, 7p, 7q, 8p, 8q, 11p, 11q, 12q, 14q, 17q, 19q, 20p, and 20q. The pattern of alterations is dominated by gains on 5p and 20q with loss of 4p, all of which were already present in a patient with carcinoma in situ. Immunohistochemical analysis of mismatch repair genes MLH1, PMS2, MSH2, and MSH6 showed negative immunostaining in 1 neoplasm (5%). MSI of BAT25 and BAT26 was seen in 3 tumors (14%) including the neoplasm with aberrant immunostaining. In conclusion, we constructed a genomic profile of DALM/UCC including several novel genetic alterations. Further, we found a low percentage of MSI. Thus, DALM/UCCs display profound chromosomal instability, but this is not associated with concurrent MSI.

Array comparative genomic hybridization, expression array, and protein analysis of critical regions on chromosome arms 1q, 7q, and 8p in adenocarcinomas of the gastroesophageal junction

Survival rates of adenocarcinomas of the gastroesophageal junction (GEJ) are low, because these tumors are generally in an advanced stage by the time they are detected. Chromosomal regions 1q32, 7q21, and 8p22 display critical alterations in GEJ cancers; however, the genes underlying alterations in these genomic areas are largely unknown. To delineate overexpressed genes, we performed array comparative genomic hybridization (aCGH) and mRNA expression analysis of 15 GEJ adenocarcinoma samples using a fine-tiling cDNA array covering chromosome segments 1q31.3~q41 (193.9-215.8 Mb: 21.9 Mb), 7q11.23~q22.1 (72.3-103.0 Mb: 30.7 Mb), and 8p23.1~p21.3 (11.1-20.7 Mb: 9.6 Mb). Based on a mRNA overexpression criterion, 11 genes were selected: ELF3 and SLC45A3 on 1q; CLDN12, CDK6, SMURF1, ARPC1B, ZKSCAN1, MCM7, and COPS6 on 7q; and FDFT1 and CTSB on 8p. The protein expression levels were subsequently determined by immunohistochemical analysis of the cancer samples. There was a significant correlation between genomic amplification, mRNA, and protein expression or overexpression for CDK6, a cell cycle regulator on 7q21.2 (92.1 Mb; P<0.01); other genes showed less stringent associations. In conclusion, using a straightforward approach we constructed a targeted gene profile for GEJ adenocarcinomas.

Genomic Analysis of Early Adenocarcinoma of the Esophagus or Gastroesophageal Junction: Tumor Progression Is Associated with Alteration of 1q and 8p Sequences

Early (T1 stage) adenocarcinoma of the esophagus or gastroesophageal junction is a potentially curable disease. We analyzed the genomic spectra of 33 early neoplastic lesions after subdividing the tumors into six depths of invasion (T1–mucosal, m1–m3; T1–submucosal, sm1–sm3). Two subgroups were defined, T1m1–sm1 (n = 18) and T1sm2–sm3 (n = 15). The latter group is associated with frequent lymphatic spread and a high percentage of local and/or distant recurrence. Comparative genomic hybridization with a genomewide 3,500‐element BAC‐PAC array revealed a characteristic gastroesophageal adenocarcinoma pattern of changes, with losses on chromosome arms 4pq, 5q, 8p, 9p, 17p, and 18q and gains on 1q, 6p, 7pq, 11q, 15q, 17q, and 20pq. However, when the two groups were compared, the following BAC clones showed significantly more alterations in the T1sm2–sm3 group: RP11‐534L20 (1q32.1) and RP11‐175A4 (6p21.32), showing gains, and RP11‐356F24, RP11‐433L7, and RP11‐241P12 (all at 8p), showing losses. Gain of RP11‐534L20 (1q32.1) and loss of RP11‐433L7 (8p22) were associated not only with a recurrence‐free period (P = 0.0007 and 0.007, respectively), but also with regional lymphatic dissemination (P = 0.005 and 0.003, respectively). These DNA clones can be considered genomic markers for the aggressive behavior of early esophageal and gastroesophageal junction adenocarcinoma.

Molecular dissection of the chromosome band 7q21 amplicon in gastroesophageal junction adenocarcinomas identifies cyclin-dependent kinase 6 at both genomic and protein expression levels.

Amplification of chromosome band 7q21 has been frequently detected in various types of cancer including gastroesophageal junction (GEJ) adenocarcinomas. At present, no gene has been disclosed that can explain this frequent amplification of 7q21 in GEJ carcinomas. Therefore, a detailed genomic analysis of the 7q21 region was performed on a selected series of GEJ adenocarcinomas, i.e., 14 primary adenocarcinomas and 10 cell lines, by array comparative genomic hybridization (aCGH) with a 7q11.22‐q31.2 contig array. A distinct peak of amplification was identified at 92.1 Mb in 7q21.2, precisely comprising cyclin‐dependent kinase 6 (CDK6), a gene involved in cell cycle regulation. A smaller peak was seen at 116.2 Mb in 7q31.2, the locus of the MET proto‐oncogene. No distinct peak was detected for the hepatocyte growth factor (HGF) at 81.3 Mb in 7q21.11. An immunoprofile of HGF, CDK6 and MET revealed a strong correlation between aCGH and immunohistochemical protein expression for CDK6 (P = 0.002). Furthermore, immunohistochemistry did not show expression of CDK6 in Barretts dysplasia and carcinoma in situ, correlating expression of CDK6 with a malignant phenotype. We conclude that high‐resolution genomic analysis and immunoprofiling identify CDK6 as the main candidate target for the recurrent amplification of 7q21 in GEJ adenocarcinomas.

Genetic evaluation of the dysplasia-carcinoma sequence in chronic viral liver disease: a detailed analysis of two cases and a review of the literature.

Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies, especially in Asia and Africa, but also in the Western world its incidence is increasing. HCC is a complication of chronic liver disease with cirrhosis as the most important risk factor. Viral co-pathogenesis due to hepatitis B virus (HBV) and hepatitis C virus (HCV) infection seems to be an important factor in the development of HCC. Curative therapy is often not possible due to the late detection of HCC. Thus, it is attractive to find parameters which predict malignant transformation in HBV- and HCV-infected livers. In the past decade, preneoplastic lesions, i.e. dysplastic foci or nodules, have gained interest as possible markers for imminent malignancy. Noteworthy, dysplastic liver lesions are increasingly detected by imaging techniques. We describe here two cases of chronic viral liver disease, one HBV-and one HCV-related, in which dysplastic lesions were present adjacent to HCC. In the HBV case, a (smaller) satellite of HCC was present as well. The neoplastic specimens were investigated by comparative genomic hybridization (CGH) and in situ hybridization (ISH). Both methods revealed multiple genetic alterations in the HCCs. The genetic patterns of the HBV-related HCC and the satellite tumor showed many shared alterations suggesting a clonal relationship. A subset of genetic changes were already present in dysplasias illustrating their preneoplastic nature. Surrounding liver cirrhosis samples did not display chromosomal aberrations. A literature survey illustrates the relative paucity of information concerning genetic alterations in preneoplastic liver lesions. However, all the data strongly suggests a role for liver cell dysplasia as a precursor condition of liver cell cancer.

Genomic analysis of a case of multifocal adenocarcinoma in ulcerative colitis

Long-standing ulcerative colitis is associated with an elevated risk of developing colonic adenocarcinoma. A very limited group of patients present with multiple synchronous cancers. This could be due to either a multifocal presentation of the same neoplastic clone or different tumors arising in a large area of polyclonal dysplastic colonic mucosa (“field cancerization”). Here, we describe a patient with long-standing colitis and three different tumors in the rectosigmoid part of the large bowel. Clonal evaluation of the lesions was performed by array-based comparative genomic hybridization. These three neoplasms showed a comparable pattern of genomic alterations characterized by gains of chromosomes 12, 13, and 20. Noteworthy, dysplastic mucosa distal to the three cancers displayed a completely different pattern of genomic changes indicating that different cell lineages were present. In addition, all three carcinomas were microsatellite stable and revealed identical immunoprofiles for several cancer-associated genes. We conclude that these three multifocal tumors must have originated from the same preneoplastic lineage.