Loukas Kaklamanis

Molecular analysis of APC mutations in familial adenomatous polyposis and sporadic colon carcinomas

Mutations in the APC gene give rise to familial adenomatous polyposis (FAP) and also occur in many, perhaps most, sporadic colon cancers. By screening with single-strand conformation polymorphism analysis we identified several mutations in a small region of the APC gene in both FAP and sporadic cancers. These mutations were either point mutations or small deletions or insertions causing frameshifts, and all generated stop codons. One 5 base-pair deletion was found in a sporadic colon tumour, a colorectal cancer cell line derived from a sporadic colon tumour, and in four unrelated FAP patients. This mutation produces distinctive heteroduplex bands, which can be detected with a simple non-radioactive assay. Our findings suggest that highly localised short sequences, essentially runs that code for adenine and thymine, may account for up to 20% of all observed APC mutations.

Up-regulation of macrophage wnt gene expression in adenoma-carcinoma progression of human colorectal cancer.

SummaryDefects in the APC-β-catenin pathway are common in colon cancer. We investigated whether aberrant regulation of upstream ligands stimulating this pathway occur in colon cancer. Using RNAase protection analysis, six out of eight wnt genes were expressed in 14 matched cases of normal, adenomatous and malignant colorectal tissues. Wnt 2 and wnt 5a were significantly up-regulated in the progression from normal through adenoma to carcinoma. Transcripts for wnts 4, 7b, 10b and 13, but not wnt 2 and wnt 5a were detected in several colorectal cell lines. In situ hybridization demonstrated that wnt 2 and wnt 5a transcripts were mainly in the lamina propria/stroma region with labelling predominantly in macrophages. Immunostaining with CD68 confirmed the wnt-expressing cells as macrophages. These results show a major difference in wnt expression in colon cancer compared to colon adenomas and suggest stromal wnt expression may play a role in tumour progression.

Selection for β2-microglobulin mutation in mismatch repair-defective colorectal carcinomas

Novel peptide antigens complexed with human leukocyte antigen (HLA) and beta 2-microglobulin (beta 2M) molecules are presented at the cell surface to cytotoxic T lymphocytes (CTLs), provoking lysis of the antigen-presenting cell [1]. In tumor cells, genetically altered or abnormally expressed proteins provide a source of peptides that can be presented to CTLs; the resulting anti-tumour CTL responses may provide part of the bodys defence against cancer. Disabling mutations in the HLA and beta 2M proteins required for peptide presentation allow a tumour cell to escape destruction by CTLs. Cells with deficient DNA mismatch repair have high spontaneous mutation rates [2] and produce many altered proteins that are a potential source of numerous unique peptides. Mutator tumour cells might therefore be particularly vulnerable to immune surveillance and CTL attack. Mutator phenotypes [3,4] and loss of beta 2M (or HLA) expression [5,6] are both relatively common among sporadic colorectal tumours. We have compared the frequency of beta 2M mutations in sporadic colorectal and other tumours with and without a mutator phenotype. Mutations were more frequent among colorectal tumours with the microsatellite instability indicative of a defect in DNA mismatch repair. The inactivating beta 2M mutations were predominantly frameshifts, which is consistent with the underlying mismatch repair defects. Evasion of immune surveillance by acquiring beta 2M mutations therefore occurs at high frequency in tumour cells with a mutator phenotype due to defective DNA mismatch repair.

EARLY EXPRESSION OF bcl-2 PROTEIN IN THE ADENOMA–CARCINOMA SEQUENCE OF COLORECTAL NEOPLASIA

bcl‐2 was originally identified as an oncogene involved in follicular lymphomas as a result of chromosomal translocation (14;18). It is now believed that bcl‐2 is implicated in the regulation of cell death by inhibiting apoptosis and that its expression is not restricted to haematopoietic cells, but is also present in many epithelia and mesenchymal tissues. Recent studies have analysed the expression of this molecule in a variety of non‐lymphoid malignancies including lung, breast, prostate, and nasopharyngeal carcinomas and neuroblastoma. In the present study, 50 colorectal adenomas, 10 hyperplastic polyps, and 142 carcinomas, including 25 arising from pre‐existing adenomas, were examined using an antibody detecting the bcl‐2 protein product. In non‐neoplastic mucosa, bcl‐2 was expressed in the crypt cells only, whilst the more differentiated surface epithelial cells lacked any demonstrable bcl‐2. Forty‐one of the 50 adenomas (82 per cent) and 48 of the 142 carcinomas were positive for bcl‐2 expression. All hyperplastic polyps were negative. A reciprocal relationship was found between bcl‐2 reactivity and p53 overexpression, as detected by DO7 antibody, in approximately 65 per cent of the cases. The bcl‐2‐positive/p53‐negative subgroup showed a strong correlation (P=0·0056) with negative lymph node status (Dukes A and B), implying a less aggressive pathway of neoplastic transformation.

Bcl-2 protein expression: association with p53 and prognosis in colorectal cancer.

Bcl-2 expression in colorectal carcinomas was studied in a series of 224 patients and the relation to p53 expression, stage and survival assessed. Bcl-2 expression was down-regulated compared with normal mucosa in 67% (151) of the cases. In 144 cases staining was positive for p53 (MAB DO7), and 41 of these 144 p53-positive cases were also bcl-2 positive (28%) compared with 32 of the remaining 80 p53-negative cases (40%). Survival was significantly worse (P = 0.01) in the p53-positive cases. Bcl-2-positive cases, including patients in all Dukes stages, had a slightly better prognosis which was not statistically significant. However, cases at an early stage (Dukes stages A and B) and with negative p53 status, had a much better prognosis if they showed bcl-2 protein expression, suggesting that the bcl-2 status itself has an effect on prognosis (P = 0.01). Neither bcl-2 nor p53 alone was correlated with stage, but when examined by both p53 and bcl-2 status a group [bcl-2(+)/p53(-)] with better prognosis was defined. The last group was significantly lower Dukes stage, with 26 out of 32 cases (81%) being A or B compared with 22 (11%) of the 202 remaining cases (P = 0.004). Thus, either loss of bcl-2 expression or gain of abnormal p53 expression is associated with high stage and poor prognosis. The bcl-2(+)/p53(-) phenotype is similar to that of normal mucosa, and these results suggest that such cases represent an indolent group at an early stage in the progression of colorectal cancer.

The expression of the B‐cell marker mb‐1 (CD79a) in Hodgkin's disease

Recent evidence indicates that membrane‐bound immunoglobulin on B lymphocytes is associated with a molecule which comprises the products of the mb‐1 and B29 genes. This molecule is a highly specific marker for B‐cells, presumably because of its central functional role in antigen triggering, and has recently been clustered as CD79a at the 5th Leucocyte Workshop. Recently there has been controversy surrounding reports of B‐cell antigen expression by Reed–Sternberg and related cells, and we have therefore studied 108 cases of Hodgkins disease immunohistochemically using a novel antibody which detects mb‐1 protein in paraffin sections. The results were compared with those achieved using antibody L26 to detect CD20. The mb‐1 protein was present in the neoplastic cells in all 14 cases of lymphocyte predominance Hodgkins disease studied, and CD20 immunoreactivity was also found in seven of the eight cases of this subtype studied. Of the non‐lymphocyte predominance cases, 20% (19/94) expressed mb‐1 and 30% (20/67) CD20 in the Reed–Sternberg cells, but the cells positive for either of these two markers usually constituted only a very small proportion of the neoplastic population. However, in occasional cases (one of 94 for mb‐1 and five of 67 for CD20), more than 50% of the neoplastic cells expressed one or both B‐cell antigens. These results confirm the B‐cell origin of the neoplastic cells in lymphocyte predominance Hodgkins disease, but they also indicate that, contrary to our previous study, mb‐1 expression may occasionally be found in what appears, on histological grounds, to be other types of Hodgkins disease.

Nuclear localization of human AP endonuclease 1 (HAP1/Ref-1) associates with prognosis in early operable non-small cell lung cancer (NSCLC)

The present study examined the immunohistochemical expression of human AP endonuclease 1 (HAP1/Ref‐1), the major endonuclease in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA, in normal lung and lung carcinomas. Cellular expression of HAP1 was determined using a standard avidin–biotin–peroxidase complex (ABC) technique and an anti‐HAP1 rabbit polyclonal antibody on paraffin‐embedded tissue sections from normal lung and in 103 primary non‐small cell lung carcinomas (NSCLCs). In normal lung, the staining for HAP1 was found to be both nuclear and cytoplasmic in the pneumocytes of the alveoli. Superficial ciliated cells of the bronchial epithelium presented cytoplasmic staining, while staining for the basal cells was mostly nuclear. Bronchial glandular cells demonstrated mixed nuclear and cytoplasmic staining. Lung carcinomas showed all patterns of expression for HAP1. Loss of HAP1 expression was associated with low proliferation index ( p=0·01) and with squamous histology ( p=0·04). In squamous carcinomas, a significant correlation was observed between positive nuclear HAP1 and negative p53 expression ( p=0·03). A survival benefit was seen in patients presenting nuclear HAP1 expression and those presenting the nuclear HAP1+/p53− phenotype ( p=0·01 and 0·007, respectively). It is concluded that nuclear HAP1 localization may be relevant to its role as a DNA repair protein and/or to the recently proposed role as an activator of wild‐type p53, and thus to the better outcome seen in this group of patients. Copyright

The epidemiologic profile of Kaposi's sarcoma in Greece prior to and during the AIDS era

To determine the incidence rates and to describe the epidemiological patterns of non‐AIDS Kaposis sarcoma in the central southern area of Greece during the period 1974–1989, all 473 incidence cases reported to Pathology Departments were studied. The mean age (SD) was 67.6 (12.9) years among 297 males and 66.1 (15.9) years among 176 females. The mean age‐standardized (Greek population 1981) incidence rate was 0.47 cases per 100,000 total population per year (males 0.62, females 0.32). The standardized incidence rates increased over time for males, with the incidence‐rate ratios relative to the earliest period, 1974–1978, being 1.44 (95% CI, 1.02–2.04) for the 1979–1983 interval and 2.12 (95% CI, 1.55–2.90) for the 1984–1989 interval. However, the rates for females did not show a similar pattern. The age‐adjusted male:female ratio was 1.6 in 1974–1983 and 2.6 in 1984–1989. Poisson‐regression modelling suggested a shift in the age‐specific incidence rate in men, towards younger ages during the last period, 1984–1989.Int. J. Cancer 70:538–541.

Loss of interleukin 4 receptor-associated molecule gp200-MR6 in human breast cancer: prognostic significance.

Several in vitro studies stress a potentially important role of interleukin 4 (IL-4) and the related gp200-MR6 molecule in the immunological response to cancer and in tumour proliferation. In the present study, we assessed the expression of gp200-MR6 in primary breast cacrinomas using the MR6 monoclonal antibody. Results were correlated with tumour parameters (T-,N-stage, histology, grade, oestrogen and epidermal growth factor (EGF) receptors), and the impact on survival was assessed. Twenty-four out of 110 cases (22%) were positive for gp200-MR6, 62 out of 110 (56%) expressed weak staining and 24 out of 114 (22%) did not stain. The normal breast epithelia were invariably stained for gp200-MR6 showing that down-regulation or loss of this molecule occurred during the evolution of breast cancer. Gp200-MR6 loss was independent from differentiation, nodal positivity and oestrogen receptor levels as well as patients age. Loss of the gp200-MR6 molecule was more frequent in lobular cases (P=0.03). The overall survival was better, although not reaching statistical significance, in patients with positive gp200-MR6 expression (92% alive at 5 years compared with 70% for those with weak or no expression, P=0.1). The local relapse-free survival was independent of gp200-MR6 status. It is concluded that loss of gp200-MR6 may be one of the mechanisms through which breast cancer cells escape immune surveillance, resulting in an increased metastatic potential and poorer outcome. Evidence of down-regulation of the gp200-MR6 molecule has implications for IL-4-linked toxin therapy and, as IL-4 is an inhibitor of breast epithelial growth, may represent loss of a tumour-suppression mechanism.

Detection of p53 in Hodgkin's disease using the monoclonal antibody PAb248

The recent demonstration that the murine anti‐p53 monoclonal antibody PAb248 can identify human p53 in a variety of normal tissues proves that immunohistochemical detection does not necessarily indicate the presence of mutations. PAb248 can detect p53 protein in a cytoplasmic‐perinuclear localization, not previously described. The present study presents the expression of this antibody in a series of 34 cases of Hodgkins disease, comparing it with the antibodies CM1, PAb1801, and PAb240. In all cases, PAb248 showed uniform cytoplasmic‐perinuclear staining in small and medium‐sized lymphocytes, while it was constantly negative in Hodgkin, Reed–Sternberg (R–S/H) cells, and variants. This pattern of staining was the opposite to that observed with the antibodies CM1, PAb1801, and PAb240, where the staining was nuclear and restricted to the R–S/H cells, with the small lymphocytes being negative. p53 can be found in different conformations and localizations, with the cytoplasmic‐perinuclear localization mainly, although not exclusively, being found in normal and reactive tissues and the nuclear localization being mainly expressed by neoplastic cells. These results give further support to the theory that the R–S/H cells are the neoplastic population in Hodgkins disease, while the surrounding lymphocytes are reactive.