Cytokeratin 7 and cytokeratin 20 expression in colorectal adenocarcinomas

Cytokeratin 7 (CK7) and cytokeratin 20 (CK20) are low molecular weight cytokeratins. The expressions of CK7 and CK20 have been studied in various primary and metastatic carcinomas. Their expression patterns may help to distinguish the site of origin of metastatic carcinomas. We investigated the expressions of CK7 and CK20 in 196 cases of colorectal carcinoma. Paraffin sections of 196 colonic adenocarcinomas were randomly selected, retrieved, and immunostained for CK7 and CK20 with a standard avidin-biotin complex method. CK7 was expressed in 34/196 (17.3%) and CK20 in 159/196 (81.1) cases of colorectal adenocarcinoma. CK7−/CK20+ had the greatest proportion (65.8%) in colorectal carcinomas. The CK7+/CK20+ immunophenotype was identified in 30/196 (15.3%), CK7−/CK20− in 33/196 (16.9%), and CK7+/CK20− in 4/196 (2%) colon adenocarcinomas. The CK7 and CK20 expression patterns were different in colorectal carcinomas according to histological grade, location of the tumor, and lymph node metastasis. CK20 positivity was more common in low grade carcinomas than in high grade carcinomas (85.1% versus 47.6%) and in rectal and sigmoid carcinomas than in proximal colon carcinomas (88.2% versus 63.2% and 88.9% versus 63.2%, respectively). Furthermore, CK7 expression was more common in tumors with lymph node metastasis than in non-metastatic tumors (25.3% versus 11%). In conclusion, a considerable number of colorectal carcinomas showed reactivity to CK7 (17.3%) or no reactivity to CK20 (18.9%). Therefore, CK7 positivity or CK20 negativity does not rule out a colorectal origin of metastatic carcinoma.     

Cytogenetics of colorectal adenocarcinomas

The occurrence of nonrandom chromosomal anomalies in colorectal adenocarcinomas could be demonstrated from the cytogenetic study of 100 cases. The most frequent changes are a rearrangement of chromosome 17, leading to the loss of its short arm and a loss of one chromosome 18. Three types of tumors with abnormal karyotypes can be defined. First are the monosomic-type near-diploid tumors (MD), characterized by a monosomy of both 17p and chromosome 18 mostly associated with other recurrent monosomies. In two of three cases, one or several minor derived polyploid subclones are also observed. Second are the monosomic-type polyploid tumors (MP), which have a pattern of chromosome imbalance very similar to that of MD tumors. They derive from MD tumors by endoreduplication followed by complete disappearance of the original MD clone. Third are the trisomic-type tumors (TT), which lose either 17p or chromosome 18 or none, most of the anomalies being gains of entire chromosomes. These TT tumors never undergo endoreduplication. In addition, seven tumors with normal karyotypes were found and may constitute another category (NT). A nonrandom distribution of these tumor types in relation to tumor site was observed, since in the distal colon, TT and NT tumors are underrepresented and endoreduplications are significantly more frequent. The level of chromosomal mutagenesis is two- to threefold higher in MD and MP than in TT tumors. More than 95% of the rearrangements are unbalanced, and most of them result from breakpoints located in juxtacentromeric heterochromatin. A good correlation is found between our results and the available molecular data on allelic losses. The involvement of recessive tumor suppressor genes in colorectal tumorigenesis and the possible relationship between chromosomal imbalances and deviations in metabolic pathways is described.     

Trisomy 7 in nonneoplastic cells

The somatic mutation theory of tumorigenesis states that mutations are necessary for tumor development. On the other hand, acquired, clonal chromosomal alterations are occasionally detected in otherwise normal, nonneoplastic cells—for example, loss of sex chromosomes occurs in bone marrow cells and lymphocytes in elderly individuals—and it is therefore evident that not all mutations are by themselves sufficient for neoplasia to occur. Thus, the finding of an acquired, clonal chromosomal abnormality does not constitute proof that a lesion is neoplastic. Trisomy 7 has, as the sole clonal chromosomal aberration, been reported in a wide variety of epithelial tumor types but also in some mesenchymal and neurogenic neoplasms. It has been suggested to be a primary, i.e., tumor-initiating, abnormality in tumors of the bladder, brain, colon, kidney, lung, ovary, prostate, and thyroid. But data from cytogenetic studies of solid tumors, macroscopically normal tissue in the proximity of solid tumors, and nonneoplastic lesions now question the importance of a solitary +7 as a neoplasia-associated change. Most solid tumors in which trisomy 7 has been found as the sole change in one clone have also displayed other, cytogenetically unrelated, clones with complex karyotypic abnormalities. Such karyotypic differences among coexisting clones could indicate that the neoplasm is polyclonal, that the cytogenetically disparate clones have emerged during tumor progression from one original clone carrying submicroscopic genomic changes only, or that the clone with +7 does not represent the tumor parenchyma. The latter interpretation is supported by the finding of cells with trisomy 7 in macroscopically normal tissue outside tumors of the brain, kidney, and lung. A seemingly decisive argument against the belief that the finding of an acquired, clonal +7 proves that a neoplasm exists is the detection of clones with an extra copy of chromosome 7 in several nonneoplastic lesions and tissues, such as atherosclerotic plaques, chorionic villi, chronic pyelonephritis, Dupuytren's contracture, focal steatosis of the liver, Peyronie's disease, and rheumatoid synovitis. That the abnormality arises in vivo has been shown by in situ hybridization with chromosome 7 specific probes; +7 is no in vitro artifact. It is unknown in which cell type the trisomy occurs; some data from colon adenomas favor epithelial cells, whereas data from kidney tumors and colon carcinomas suggest that the +7 arises in cells of the immune system. This question may possibly be resolved by obtaining a pure cell line with trisomy 7 cells only, to be characterized further by immunologic and morphologic methods. Another line of investigation might be to search for clonal chromosomal abnormalities in nonneoplastic tissues in other species. Finally, it remains to be elucidated whether +7 is a neutral genome mutation or whether it confers a selective advantage upon the cell. © 1993 Wiley-Liss, Inc.     

Histological inhomogeneity of colorectal adenocarcinomas

The histological inhomogeneity of colorectal adenocarcinomas has hardly been systematically investigated up to now. Currently, however, this awareness is gaining in importance, e.g. within the framework of grading measures, for analysis of immunohistochemical results or for interpretation of flowcytometric data. Thus, 100 colon carcinomas each were semiquantitatively investigated on four different sites in terms of either homogeneous or inhomogeneous expression of histological, cytological and immunohistochemical (CEA, UEA I) criteria. Although all carcinomas contained tubular structures, there was no intraindividual uniformity but considerable inhomogeneity on the part of histological as well as cytological features. Quantitative and qualitative positivity of CEA and UEA I too, varied markedly. Our results lead to the conclusion that the phenotypic inhomogeneity of colorectal carcinoma is at least partially attributable to genetic tumor cell instability. In spite of this, there might be a trend to intra-individual localisation-related tumor cell differentiation, because several parameters showed significant differences between luminal and deep carcinoma regions.     

Karyotypic characterization of colorectal adenocarcinomas

Cytogenetic analysis of short-term cultures from 52 primary colorectal adenocarcinomas revealed clonal chromosome aberrations in 45 tumors, whereas the remaining 7 had a normal karyotype. More than 1 abnormal clone was detected in 26 tumors; in 18 of them, the clones were cytogenetically unrelated. The modal chromosome number was near-diploid in 32 tumors and near-triploid to near-tetraploid in 13. Only numerical aberrations were identified in 13 carcinomas, only structural aberrations in 3, and 29 had both numerical and structural changes. The most common numerical abnormalities were, in order of decreasing frequency, gains of chromosomes 7, 13, 20, and Y and losses of chromosomes 18, Y, 14, and 15. The structural changes most often affected chromosomes 1, 17, 8, 7, and 13. The most frequently rearranged chromosome bands were, in order of decreasing frequency, 13q10, 17p10, 1p22, 8q10, 17p11, 7q11, 1p33, 7p22, 7q32, 12q24, 16p13, and 19p13. Frequently recurring aberrations affecting these bands were del(1)(p22), i(8)(q10), i(13)(q10), and add(17)(p11–13). The most common partial gains were from chromosome arms 8q, 13q, and 17q and the most common partial losses from chromosome arms 1p, 8p, 13p, and 17p. A correlation analysis between the karyotype and the clinicopathologic features in our total material, which consists of altogether 153 colorectal carcinomas, including 116 with an abnormal karyotype, showed a statistically significant association (P < 0.05) between the karyotype and tumor grade and site. Carcinomas with structural chromosome rearrangements were often poorly differentiated; well and moderately differentiated tumors often had only numerical aberrations or normal karyotypes. Abnormal karyotypes were more common in rectal carcinomas than in carcinomas situated higher up. Near-triploid to near-tetraploid karyotypes were more than twice as frequent in tumors of the distal colon as in those of the proximal colon and rectum. The cytogenetic data indicate that carcinomas located in the proximal colon and rectum, which often are near-diploid with simple numerical changes and cytogenetically unrelated clones, probably arise through different mechanisms than do tumors located in the distal colon, which more often have complex near-triploid to near-tetraploid karyotypes.     

Expression of beta-catenin in prostatic adenocarcinomas: a comparison with colorectal adenocarcinomas

We studied 101 prostatic adenocarcinomas (72 acinar 29 ductal) and 16 cases with high-grade prostatic intraepithelial neoplasia (HPIN) immunohistochemically for the expression of beta-catenin and compared the staining patterns with those of nonneoplastic prostatic epithelium and 24 colorectal adenocarcinomas. While nuclear staining for beta-catenin was evident in 20 (83%) colorectal adenocarcinomas, predominantly, membranous staining was observed in 89 prostatic adenocarcinomas (88%); the remaining 12 cases showed no immunoreactivity. In prostatic tumors expressing beta-catenin, staining intensity was comparable, increased, and decreased in 81, 4, and 4 cases, respectively compared with adjacent nonneoplastic prostatic epithelium. No prostatic adenocarcinomas demonstrated nuclear staining. The beta-catenin staining characteristics in HPIN were comparable to those in nonneoplastic prostatic epithelium. Negative staining for cytokeratins (CKs) 7 and 20, high-molecular-weight (HMW) CK, and villin and positive staining for prostate-specific antigen (PSA) were seen in 22 prostatic adenocarcinomas examined, in contrast with colorectal adenocarcinomas, which stained positively for CK20 and villin and negatively for CK7, HMWCK, and PSA. These data suggest that the beta-catenin signaling pathway acts differently in prostatic than in colorectal tumorigenesis. The immunophenotypes documented herein also might aid in the distinction between prostatic and colorectal origins when metastasis is encountered.     

Trisomy 7 and Potter syndrome

A patient with mosaic trisomy 7 and features of Potter syndrome is described. The patient was the product of a 35-week gestation and survived fourteen hours, demise being attributed to respiratory insufficiency. Autopsy confirmed pulmonary hypoplasia and renal agenesis. Additional findings included malformed, low-set ears, a flattened nasal bridge, redundant nuchal skin, positional deformation of the extremities, rocker-bottom feet, and clitorimegaly. Cytogenetic study of peripheral blood and skin fibroblast culture revealed mosaicism for full trisomy 7, the skin showing 12% of the cells to have an extra 7. Comparison with one previously confirmed case of trisomy 7 and two cases of trisomy C suggests a correlation between trisomy 7 and Potter syndrome.     

Aberrant pattern of the cytokeratin 7/cytokeratin 20 immunophenotype in colorectal adenocarcinomas with BRAF mutations

Cytokeratin 7 (CK7) and 20 (CK20) are used for the differential diagnosis of metastases from colorectal carcinomas (CRC), which are usually CK7-/CK20+, and other tumors. In our study, we performed immunohistochemical staining with CK7 and CK20 in 52 randomly selected cases of CRC and analyzed microsatellite instability status and BRAF mutations to identify those factors that may determine the changing pattern of CK7/CK20 immunophenotype in these tumors. CK7 was negative in all microsatellite stable tumors (MSS), but all carcinomas presenting microsatellite instability (MSI) and BRAF mutations were diffusely positive for this marker. CK20 was diffusely expressed in 79.06% of MSS tumors. Regarding MSI, in case with no BRAF mutations, a progressive decrease in CK20 expression was noted, and in BRAF-mutated adenocarcinomas, no expression of CK20 was observed. It seems that in case of MSI located on the proximal colon, which also presents BRAF mutations, CK20/CK7 may present a changing immunophenotype pattern, which may complicate the differential diagnosis of metastatic tumors. This is the first reported study of the relationship between CK20/CK7 immunophenotype, BRAF mutations and microsatellite status in CRC.     

Ossification in lung metastases of primary colorectal adenocarcinomas

Ossification of lung tissue is a rare phenomenon, which can be found in association with carcinoid tumors of the lung or pulmonary blastomas. Very rarely, ossifications are observed in lung metastases of extrathoracal epithelial tumors. In these cases, the most probable primary focus is a colorectal adenocarcinoma. Our question was, whether ossifications in lung metastases are pathognomonic for colorectal adenocarcinomas and how they can be pathogenetically arranged. A total of 15 lung metastases with ossifications from 5 patients suffering from a colorectal adenocarcinoma were examined by means of immunohistochemistry. Thereby, we found an increased expression of bone morphogenetic protein (BMP) 2/4 and osteonectin in tumor cells, as well as an increased stromatogenous expression of collagen type III. We conclude that there is strong evidence of a primary colorectal adenocarcinoma when ossifications in lung metastases are found. In these cases, a common metastatic spread of tumor cells and tumor stroma seems to be probable.     

Characterization of mutator pathway in younger-age-onset colorectal adenocarcinomas

The high-frequency microsatellite instability (MSI-H) phenotype, frequently identified in hereditary nonpolyposis colorectal cancer (HNPCC), also accounts for approximately 15% of sporadic colorectal cancers. Microsatellite instability (MSI) occurs from the mutational inactivation of the DNA mismatch repair genes, i.e. hMSH2 and hMLH1 in HNPCC, as well as from epigenetic inactivation of hMLH1 in sporadic colorectal tumors. The mutator pathway including microsatellite instability, hMLH1 promoter methylation, and hMSH2 and hMLH1 mutation patterns were identified in 21 sporadic colorectal adenocarcinoma patients younger than 30 yr excluding HNPCC. More than half of tumors showed MSI, with five MSI-H and six MSI-L (low-frequency microsatellite instability). Three of six MSI-H tumors showed the hMLH1 promoter methylation and did not express the hMLH1 protein. On the other hand, all MSI-L and all MSS (microsatellite stable) tumors expressed both hMSH2 and hMLH1 proteins. Two novel mutations, i.e. a missense mutation in hMLH1 and a splice-site alteration in hMSH2, were identified in two patients respectively. Although mutator pathway was implicated in younger-age-onset colorectal carcinogenesis, many tumors appeared to evolve from different genetic events other than hMSH2 and hMLH1 mutations frequently identified in HNPCC.     

Cytogenetic and molecular approaches of polyploidization in colorectal adenocarcinomas

We present the cytogenetic analysis of 23 cases of polyploid colorectal adenocarcinomas. We took advantage of the high intratumoral heterogeneity of the karyotypes to identify clones, subclones, and cell-to-cell variations. This allowed us to reconstruct the chromosomal evolution of each tumor and to propose a schema of the chromosomal changes in relation to the endoreduplication process. All but one case were characterized by a relative deficiency of chromosomes 17p and 18. Other deficiencies affecting the late-replicating X, and to a lesser degree, 1p, 5q, 14, 15, 8p, 10, 21, and 4, and excesses affecting the early-replicating X, 8q, 13, 16, 17q, and 11 were frequently associated. This pattern of imbalances is very similar to that of the monosomic type previously described in near-diploid tumors. The pattern of the 23rd tumor corresponded to those of the trisomic type tumors. These data largely confirm the existence of two distinct processes of chromosomal evolution in colorectal adenocarcinomas, with a strong tendency to undergo endoreduplication for the monosomic type near-diploid tumors. To correlate cytogenetic and molecular data, allelic losses analyses were investigated for probes of chromosomes 17p and 18. In all 12 informative tumors, a loss of heterozygosity for probes of the short arm of chromosome 17 indicated the occurrence of a rearrangement of chromosome 17 before the endoreduplication. The same was true for allelic losses for probes of chromosome 18 found in 11 of 12 informative tumors. The correlation between cytogenetic and molecular data is thus excellent and indicates that losses of 17p and 18 are early events in the tumor process.     

DNA flow cytometry of endoscopically examined colorectal adenocarcinomas

The purpose of the study was to evaluate the correlation of DNA-ploidy of colorectal adenocarcinomas (adk) with histological and clinical parameters including the survival of the patients. Multiple biopsies from 95 adk were taken during colonoscopy prior to surgery. The samples were used to obtain nuclei suspensions for specific staining of DNA content and high resolution flow cytometry. DNA-aneuploidy, i.e. the presence of more than one G0/G1 peak, was detected in 67/95 cases (71%). The individual-specific control mucosa was DNA-diploid in all cases. The mean fraction of S-phase cells was 7.2% in control mucosa and 13.6% in adk. DNA-ploidy did neither correlate with Dukes' stage nor with differentiation degree. Among the patients studied for the correlation of DNA ploidy with survival for a period extending to 30 months (n = 51), the DNA aneuploid group was estimated to be about 5 times as risky as the DNA diploid group with respect to the odds of dying. We conclude that DNA flow cytometry of colorectal adk may predict clinical outcome and be helpful in addition to histopathology.     

Increase of a urokinase receptor-related low-molecular-weight molecule in colorectal adenocarcinomas

Proteolytic activity is important for tumor growth and metastasis. Plasminogen and urokinase-type plasminogen activator (u-PA) constitute one of the most extensively studied proteolytic systems believed to participate in these processes. u-PA cleaves plasminogen to plasmin, which in turn degrades surrounding extracellular matrix and allows tumor cells to migrate to other areas. The specific receptor for u-PA (u-PAR) has also been implicated as an essential modulator in this pathway. Eleven paired samples of colorectal cancers and normal mucosal tissues from the same patients were removed at surgery. The tissues were homogenized and the supernatants assayed for u-PAR immunoreactivity, u-PAR antigen concentration, u-PAR binding activity and u-PA activity. Immunoblot analysis showed that a major u-PAR species of 55 kDa was present in all tissues. In addition, a protein band of 4 kDa, which crossreacted with anti-u-PAR antibodies, was also found in the tumors. This protein band was either absent, or present in relatively small amounts in the normal colorectal tissues. Cross-linking experiments showed that the 55 kDa band only, and not the 41 kDa band, was able to bind either single chain urokinase-type plasminogen activator (scu-PA) or the amino terminal fragment of urokinase (ATF). The tumor samples also exhibited highly elevated u-PA activity and u-PAR antigen relative to the corresponding normal tissues. Elevated u-PA activity appeared to correlate with elevated u-PAR antigen in colorectal cancers, but not in the normal tissues. These increases were also associated with increase of the u-PAR-related, low-molecular-weight protein in the tumor samples. The measurement of u-PAR and the u-PAR-related protein, in addition to u-PA activity, could have diagnostic or prognostic value in this type of cancer.     

Impaired expression of acyl-CoA synthetase 5 in sporadic colorectal adenocarcinomas

Several pathways of fatty acid metabolism have been shown to be associated with the pathogenesis of colorectal cancer. Fatty acid acyl-CoA thioesters are formed from free fatty acids and coenzyme A by the activity of acyl-CoA synthetases (ACSs). Whilst an increase in ACS4 expression has been associated with colorectal carcinogenesis, little is known about possible pathogenetic functions of other ACS isoforms, such as ACS5, in tumourigenesis. In the present study, gene expression, protein synthesis, and enzymatic activity of ACS5 in sporadic colorectal adenocarcinomas, adenomas, and established cell lines were analysed using RT-PCR, western blot analysis, immunofluorescence, and an enzymatic assay. Enhanced expression of ACS5 mRNA and protein as well as enzymatic activity was found in adenomas and in 11 (73%; group 1) of 15 colorectal adenocarcinomas investigated, while a decrease of ACS5 was seen in four tumours (27%; group 2). However, basal ACS5 enzymatic activity was increased as a percentage of the total activity of ACSs in both groups, arguing for an absolute (group 1) or relative (group 2) increase in ACS5 enzymatic activity in all adenocarcinomas investigated. These findings are reflected by in vitro analysis of three established colorectal adenocarcinoma cell lines, in which activity of ACS5 occurred. The results suggest the involvement of ACS5 in the early genesis of colorectal cancer, most likely by modification of the transport and pool formation of long-chain acyl-CoA thioesters, as recently demonstrated for other isoforms of the ACS family.     

Trisomy 7 in synovial fibroblasts obtained from arthritic joints

Inflammation Research -