Predictors for neoplastic progression in patients with Barrett's Esophagus: a prospective cohort study.

OBJECTIVES: Patients with Barrett's esophagus (BE) have an increased risk of developing esophageal adenocarcinoma (EAC). As the absolute risk remains low, there is a need for predictors of neoplastic progression to tailor more individualized surveillance programs. The aim of this study was to identify such predictors of progression to high-grade dysplasia (HGD) and EAC in patients with BE after 4 years of surveillance and to develop a prediction model based on these factors. METHODS: We included 713 patients with BE (≥ 2 cm) with no dysplasia (ND) or low-grade dysplasia (LGD) in a multicenter, prospective cohort study. Data on age, gender, body mass index (BMI), reflux symptoms, tobacco and alcohol use, medication use, upper gastrointestinal (GI) endoscopy findings, and histology were prospectively collected. As part of this study, patients with ND underwent surveillance every 2 years, whereas those with LGD were followed on a yearly basis. Log linear regression analysis was performed to identify risk factors associated with the development of HGD or EAC during surveillance. RESULTS: After 4 years of follow-up, 26/713 (3.4%) patients developed HGD or EAC, with the remaining 687 patients remaining stable with ND or LGD. Multivariable analysis showed that a known duration of BE of ≥ 10 years (risk ratio (RR) 3.2; 95% confidence interval (CI) 1.3-7.8), length of BE (RR 1.11 per cm increase in length; 95% CI 1.01-1.2), esophagitis (RR 3.5; 95% CI 1.3-9.5), and LGD (RR 9.7; 95% CI 4.4-21.5) were significant predictors of progression to HGD or EAC. In a prediction model, we found that the annual risk of developing HGD or EAC in BE varied between 0.3% and up to 40%. Patients with ND and no other risk factors had the lowest risk of developing HGD or EAC (<1%), whereas those with LGD and at least one other risk factor had the highest risk of neoplastic progression (18-40%). CONCLUSIONS: In patients with BE, the risk of developing HGD or EAC is predominantly determined by the presence of LGD, a known duration of BE of ≥10 years, longer length of BE, and presence of esophagitis. One or combinations of these risk factors are able to identify patients with a low or high risk of neoplastic progression and could therefore be used to individualize surveillance intervals in BE.

Eosinophilic oesophagitis: an enigmatic, emerging disease.

Eosinophilic oesophagitis is a disease that has emerged in recent years. It is often associated with dysphagia and oesophageal food impaction in adults. The disease is characterised by infiltration of eosinophilic granulocytes into the oesophageal mucosa. This infiltrate may be responsible for the subtle peristaltic abnormalities that can be found in these patients. Endoscopic findings are usually absent or nonspecific, although a discrete circular ring pattern of the mucosa may be noticed. Occasionally, overt endoscopic abnormalities (such as exudative changes and shearing of the mucosa) can be found. The presence of at least 15 intraepithelial eosinophilic granulocytes per high-power field in random biopsies from the whole length of the oesophagus is considered to be diagnostic. Gastro-oesophageal reflux needs to be excluded as it may lead to eosinophilic infiltration as well. Adequate diagnosis is relevant for treatment and the prevention of unnecessary further investigations. The disease responds well to the ingestion of fluticasone propionate and its long-term prognosis is generally good. But when fluticasone is discontinued recurrent symptoms are common, and some cases are severe, needing treatment with systemic corticosteroids.

Rectal carcinoma treated with short-term preoperative radiotherapy followed by abdominoperineal resection. Significantly more presacral abscesses with absence of local recurrence.

BACKGROUND: Treatment of choice for rectal carcinoma is short-term preoperative radiotherapy (5 x 5 Gy) followed by a total mesorectal excision (TME) of the rectum. This treatment has led to a reduction in local recurrence 2 years after surgery from 8.2 to 2.4%. Side effects of this treatment seem to be marginal and of no consequence. After introduction of short-term preoperative radiotherapy we noticed a rise in the postoperative presacral abscess formation which is difficult to treat and results in readmissions and prolonged hospital stay. Research was needed to investigate whether short-term preoperative radiotherapy can be held accountable for the presumed rise in presacral abscess formation in the treatment of rectal carcinoma. METHODS: A retrospective study was performed over the period January 2000-October 2004. Two groups were formed. Group 1 existed of 30 patients who underwent a TME of the rectum without short-term preoperative radiotherapy. Group two existed of 35 patients who underwent a TME of the rectum with short-term preoperative radiotherapy. RESULTS: Statistical analysis showed a significant increase in presacral abscess formation (13 vs. 40%) after introduction of short-term preoperative radiotherapy. Radiotherapy proved to be an important risk factor. Reduction in incidence of local recurrence was not evident. CONCLUSION: We found a significant increase in presacral abscess formation strongly suggestive due to the introduction of short-term preoperative radiotherapy in the treatment of rectal carcinoma. We noticed no reduction in incidence of local recurrence. We advocate that additional research is needed in order to formulate extra patient selection criteria for the use of preoperative radiotherapy in the treatment of rectal cancer.

Clinical relevance of apoptotic markers in breast cancer not yet clear.

Currently, the mainly used characteristics to predict outcome or treatment response in patients with breast cancer are tumor size, N-status, histological grade and receptor status (ER/PgR). However, these conventional clinico-pathological characteristics are of limited value. More accurate determinators are needed to select patients who are most likely to benefit from treatment in terms of prognosis as well as treatment response. Proliferation and apoptosis are assumed to play a key role in tumor progression as well as response to treatment. Currently, an increasing number of molecular factors controlling apoptosis as well as proliferation is known. The clinical relevance of apoptotic tumor markers in the treatment strategy of patients with breast cancer is the subject of this review. In addition, potential future developments are discussed.

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Evaluation of a method that supports pathology report coding.

OBJECTIVES: The paper focuses on the problem of adequately coding pathology reports using SNOMED. Both the agreement between pathologists in coding and the quality of a system that supports pathologists in coding pathology reports were evaluated. METHODS: Six sets of three pathologists each received a different set of 40 pathology reports. Five different SNOMED code lines accompanied each pathology report. Three pathologists evaluated the correctness of each of these code lines. Kappa values and values for the reliability coefficients were determined to gain insight in the variance observed when coding pathology reports. The system that is evaluated compares a newly entered report, represented as a multi-dimensional word vector, with reports in a library, represented in the same way. The reports in the library are already coded. The system presents the code lines belonging to the five library reports most similar to the newly entered one to the pathologist in this way supporting the pathologist in determining the correct codes. A high similarity between two reports is indicated by a large value of the inproduct of the vector of the newly entered report and the vector of a report in the library. RESULTS: Agreement between pathologists in coding was fair (average kappa of 0.44). The reliability coefficient varied from 0.81 to 0.89 for the six sets of pathology reports. The system gave correct suggestions in 50% of the reports. In another 30% it was helpful for the pathologists. CONCLUSIONS: On the basis of the level of the reliability coefficients it could be concluded that three pathologists are indeed sufficient for obtaining a gold standard for evaluating the system. The method used for comparing reports is not strong enough to allow fully automatic coding. It could be shown that the system induces a more uniform coding by pathologists. An evaluation of the incorrect suggestions of the system indicates that the performance of the system can still be improved.

Evidence for a bias toward intracellular antigens in the local humoral anti-tumor immune response of a colorectal cancer patient revealed by phage display.

Many patients with colorectal carcinoma (CRC) mount a cellular as well as a humoral immune response to the tumor. To investigate the nature and specificity of the humoral immune response in a CRC patient, lymphocytes infiltrating the primary colorectal tumor and lymph nodes draining the tumor were used as antibody variable (V)-gene pools for the construction of phage antibody repertoires. These libraries were first validated by selection on the antigen tetanus toxoid and shown to contain antibodies that were probably derived from both naive and memory B cells. The repertoires were then screened for the presence of antibodies directed to CRC by selection on the cell line CaCo2. For comparison, the same selections were performed with a phage antibody repertoire made from B cells of healthy donors. Striking differences were observed in the panel of specificities selected from these different repertoires: although a large panel of antibodies reactive with patient-derived primary tumors was obtained from the immune repertoires, none of these discriminated between normal colonic epithelium and colon cancer and none were reactive with cell-surface antigens. However, selections using the non-immune library did result in numerous antibodies that recognized cell surface markers on CaCo2. These data suggest a bias in the local humoral immune response in this CRC patient, directed primarily toward intracellular epithelial-cell specific target antigens.

The morphological effects of a radio frequency lesion adjacent to the dorsal root ganglion (RF-DRG)--an experimental study in the goat.

Radiofrequency (RF) lesions adjacent to the dorsal root ganglion (DRG) are increasingly used in the treatment of intractable chronic pain of spinal origin. Opinions differ on which nerve fibres are affected by these lesions. Morphological studies have been carried out to assess the effects of radiofrequency lesions on nervous tissue. Interpretation has been difficult, since most studies have been performed under circumstances which are not comparable to the clinical situation. This study was undertaken to investigate morphological effects of RF lesions as they develop in the normal clinical situation. In two goats 22 G 100 mm SMK electrodes with a 5 mm active tip (Radionics) were positioned posterior to the lumber dorsal root ganglia (DRG). Sixty-second 67;C lesions were made on one side. The contralateral side was used as sham operation. The goats were sacrificed 2 weeks after the procedure. The lesions were studied for size as well as for effects on proliferation and regeneration using Ki-67 (MIB-1). Lesions made inside the DRG (23) were 1.8-2.0 mm in size. In these lesions there was a total loss of myelinated fibres. In lesions made adjacent to the DRG there was a significantly higher MIB-1 labelling on the treated side as compared to the sham-treated side. An RF lesion inside the DRG destroys myelinated fibres. A lesion adjacent to the DRG increases MIB-1 activity, indicating proliferation and regeneration after 2 weeks, despite the fact that the lesion was made outside the ganglion.

In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody.

Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 x 10(-2) s-1. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (gamma 1) hinge region and CH3 domain. This "minibody" was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting.

Identification of colon tumour-associated antigens by phage antibody selections on primary colorectal carcinoma.

Immunotargeting of solid tumours using antibodies has become a valuable tool for the detection of cancer metastases and the treatment of minimal residual disease. However, only few tumour antigens useful for targeting have been described to date. To identify cell-surface targets on colorectal carcinoma (CRC), we selected a large, human phage antibody repertoire on freshly isolated colon tumour cells. Two antibodies were identified that reacted with epithelial cell-restricted cell-surface antigens, whereas one clone preferentially reacted with stromal cells. These antigens are tumour-associated antigens, as shown by their uniform expression in tumours of different patients and of different differentiation stages and by their limited expression on normal tissues. The pattern of reactivity in immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) suggests that these antigens are different from previously identified tumour-associated antigens (e.g. Ep-CAM or c-ERB-2). This phage antibody-based method may lead to the cloning of novel tumour antigens that are useful for the immunotargeting of solid tumours.

Cell proliferation and apoptosis in stage III inoperable non-small cell lung carcinoma treated by radiotherapy.

PURPOSE: The purpose of this study was to assess the prognostic value of the expression of p53 and bcl-2, the apoptotic index and the expression of topoisomerase II alpha in patients with inoperable non-small cell lung cancer (NSCLC) treated with high dose radiotherapy. PATIENTS AND METHODS: A number of 161 patients with inoperable NSCLC treated with high dose radiotherapy (60 Gy) were included. Immunohistochemical analysis was used to assess the expression of nuclear p53-protein, topoisomerase II alpha and cytoplasmatic expression of bcl-2, while spontaneous apoptosis was assessed using in situ labeling. The minimal follow up period was 2 years. RESULTS: Local control did not only depend on the presence of p53 expression, but also on the proportion of p53 positive cells. The most important prognostic factor was the apoptotic index. A high apoptotic index was associated with worse local control, more distant metastases and a significantly worse overall survival. No association was noted between the expression of bcl-2 and topoisomerase II alpha with any of the endpoints. CONCLUSION: This study indicates that p53 expression and the apoptotic index are prognostic factors with regard to local control in patients with inoperable NSCLC treated with radiotherapy and by combining these 2 factors, a clinically relevant estimation of the local control probability can be made. The apoptotic index turned out to be the only factor significantly related to survival.

Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning.

In various clinical studies, Hodgkin's patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the nonhuman therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics.

Morphological changes in mesothelial cells induced by shed menstrual endometrium in vitro are not primarily due to apoptosis or necrosis.

In a previous study on the pathogenesis of endometriosis, we observed that constituents of menstrual effluent induce morphological alterations in human mesothelial cells. In this study, we investigated whether these alterations were associated with apoptosis or necrosis or were the result of cellular remodelling. After overnight incubation of confluent monolayers of human omental mesothelial cells (HOMEC) with conditioned media prepared from menstrual effluent shed anterogradely, severe alterations in morphology were observed. Typical polygonal mesothelial cell cultures at confluency acquired elongated spindle morphology, resulting in gaps between the cells. In contrast, mesothelial cells from the control groups receiving culture medium only, retained a normal morphology. Immunofluorescence staining revealed that cytokeratin, vimentin and actin filaments were still present, homogeneously distributed in the cell cytoplasm following changes in morphology. To evaluate whether the morphological alterations were associated with apoptosis and/or necrosis, the cells were stained with the M30 CytoDeath antibody or annexin V with propidium iodide and analysed using flow cytometry. The results showed that only a small percentage (1-7%) of the affected HOMEC were undergoing apoptosis or necrosis. We conclude that the profoundly altered morphology of HOMEC is a result of cellular remodelling and that the role of apoptosis and necrosis is negligible. Soluble paracrine factors released by cells isolated from menstrual effluent shed anterogradely may induce a reorganization of the cytoskeleton. As a result, the underlying basement membrane will be exposed and the mesothelium may no longer prevent implantation of endometrium shed retrogradely into the peritoneum, thus facilitating the development of endometriosis.

Treatment of leptomeningeal metastases evaluated by interphase cytogenetics.

PURPOSE: Although cytologic examination of CSF is the primary method for the evaluation of response to therapy for leptomeningeal metastases (LMMs), the procedure's sensitivity decreases throughout the course of protracted therapy. We studied whether this response could be monitored more accurately through the detection of numerical chromosomal aberrations by interphase cytogenetics, using fluorescence in situ hybridization (FISH). PATIENTS AND METHODS: Seven patients treated for LMMs and with a known numerical aberration for chromosome 1 in their pretreatment CSF were included in this study. Up to 16 consecutive CSF samples were analyzed by means of the fluorescence in situ hybridization (FISH) technique for cells with aberrant chromosome 1 content. The results of routine cytology and FISH analyses were compared and were correlated with each patient's neurologic status. RESULTS: Routine cytology detected malignancies in only 24 of the 76 samples, all of which were classified as chromosomally abnormal by FISH (except for two samples that could not be evaluated). Moreover, FISH demonstrated aneusomic cells in 32 additional samples, which could therefore be classified as malignant. The FISH results correlated better with patient neurologic status in that more malignant cells were detected in the CSF of neurologically deteriorating patients. CONCLUSION: Using FISH in addition to performing routine cytologic examination of CSF led to a more accurate evaluation of response to treatment in patients treated for LMMs.

Anti-apoptotic phenotype is associated with decreased locoregional recurrence rate in breast cancer.

PURPOSE: Tumor stage and nodal status are the most important factors predicting locoregional recurrence in breast cancer. We wanted to investigate the prognostic value of some newer molecular genetic markers for the occurrence of a locoregional recurrence, in order to improve the selection of patients for locoregional adjuvant therapy. METHODS: Bcl-2, p53, MIB-1, pS2 and CD44v6 were determined immunohistochemically on formalin-fixed and paraffin embedded tumour tissues of 163 patients treated by modified radical mastectomy between 1982 and 1987. Postoperative irradiation was given to 35 patients to the intermammary chain only and to only 13 (8%) patients to the chest wall with or without the regional lymph nodes. Node-positive patients were treated with CAF adjuvant chemotherapy and were randomized for whether or no additional Medroxyprogesteroneacetate (MPA). A multivariate analysis was performed on a number of potential prognostic factors. The risk for locoregional recurrence was estimated using the competing risk approach. RESULTS: After a median period of 7.5 years 28 patients developed a locoregional recurrence. The cumulative incidence of loco-regional recurrence at 10 years was 17%. Bcl-2 and p53 were found to be independent factors predicting locoregional recurrence, whereas a trend was found for MIB-1. Increased Bcl-2 as well as p53 expression were associated with a decreased risk, whereas the increased presence of MIB-1 was associated with an increased risk. CONCLUSION: Results indicate that molecular markers of apoptosis as well as proliferation provide additional information for the risk of locoregional recurrence after modified radical mastectomy. If confirmed, these markers may play a role in the selection of appropriate locoregional adjuvant treatment after primary surgery.

Supporting the classification of pathology reports: comparing two information retrieval methods.

In this contribution two methods from the domain of information retrieval are compared. The goal of the retrieval is to select from a library of pathology reports those ones that are most similar to a given report. The SNOMED codes that accompany these reports are presented to the pathologist who has to code the given report with the aim to improve the quality of coding. The reports were represented either as a vector of words or as a vector of N-grams. Both 4-, 5- and 6-grams were used. The similarity of the reports was determined by comparing the SNOMED terms that were added to the reports. It could be concluded that the word-based method was consistently better than the N-gram method.

Aneurysm of the umbilical vein: case report and review of literature.

We report a case of aneurysm of the umbilical vein, causing fetal death at 41 weeks gestation. We conclude that these aneurysms are a complication of congenital thinning of the vessel wall and want to emphasize that in stillbirths the cause of death may only be revealed by careful placental examination, including the umbilical cord.

Guided selection of a pan carcinoma specific antibody reveals similar binding characteristics yet structural divergence between the original murine antibody and its human equivalent.

Antibody engineering provides an excellent tool for the generation of human immunotherapeutics for the targeted treatment of solid tumours. We have engineered and selected a completely human antibody to epithelial glycoprotein-2 (EGP-2), a transmembrane glycoprotein present on virtually all human simple epithelia and abundantly expressed on a variety of human carcinomas. We chose to use the procedure of "guided selection" to rebuild a high-affinity murine antibody into a human antibody, using two consecutive rounds of variable domain shuffling and phage library selection. As a starting antibody, the murine antibody MOC-31 was used. After the first round of guided selection, where the V(H) of MOC-31 was combined in Fab format with a human V(L)C(L) library, a small panel of human light chains was identified, originating from a segment of the VkappaIII family, whereas the MOC-31 V(L) is more homologous to the VkappaII family. Nevertheless, one of the chimaeric Fabs, C3, displayed an off-rate similar to MOC-31 scFv. Combining the V(L) of C3 with a human V(H) library, while retaining the V(H) CDR3 of MOC-31, clones were selected using human V(H) genes originating from the rarely used V(H)7 family. The best clone, 9E, shows over 13 amino acid mutations from the germline sequence, has an off-rate comparable to the original antibody and specifically binds to the "MOC-31"-epitope on EGP-2 in specificity and competition ELISA, FACS analysis and immunohistochemistry. In both V(L) and V(H) of antibody 9E, three germline mutations were found creating the MOC-31 homologue residue. Structural modelling of both murine and human antibodies reveals that one of the germline mutations, 53Y in V(H) CDR2, is likely to be involved in antigen binding. We conclude that, although they may bind the same epitope and have similar binding affinity to the antigen as the original murine antibody, human antibodies derived by guided selection unlike CDR-grafted antibodies, may retain only some of the original key elements of the binding site chemistry. The selected human anti-EGP-2 antibody will be a suitable reagent for tumour targeting.

Molecular interactions in the Vogelstein model of colorectal carcinoma.

For about a decade, the model proposed by Fearon and Vogelstein has been the paradigm of the genetic alterations involved in the development of colorectal carcinoma. During this time, much information has become available on the function of the key genes in this model, as well as on their interactions. This review examines the impact of this new knowledge on the Vogelstein model. It is concluded that the model as such still stands and with a few modifications could even be strengthened in that, contrary to the original proposal, the order of genetic events seems to be essential. Crucial molecular events include derangement of the Wnt- and defects in the transforming growth factor beta (TGFbeta)-signalling pathways, which exert a synergistic effect on the cell cycle. Finally, with loss of p53 function, several checks and balances are disrupted, which paves the way to gross chromosomal aberrations and aneuploidy.

A profile of differentially expressed genes in primary colorectal cancer using suppression subtractive hybridization.

As a step towards understanding the complex differences between normal cells and cancer cells, we have used suppression subtractive hybridization (SSH) to generate a profile of genes overexpressed in primary colorectal cancer (CRC). From a 35¿ omitted¿000 clone SSH-cDNA repertoire, we have screened 400 random clones by reverse Northern blotting, of which 45 clones were scored as overexpressed in tumor compared to matched normal mucosa. Sequencing showed 37 different genes and of these, 16 genes corresponded to known genes in the public databases. Twelve genes, including Smad5 and Fls353, have previously been shown to be overexpressed in CRC. A series of known genes which have not previously been reported to be overexpressed in cancer were also recovered: Hsc70, PBEF, ribophorin II and Ese-3B. The remaining 21 genes have as yet no functional annotation. These results show that SSH in conjunction with high throughput screening provides a very efficient means to produce a broad profile of genes differentially expressed in cancer. Some of the genes identified may provide novel points of therapeutic intervention.