Background: It is widely accepted that the adenoma±carcinoma sequence represents the process by which most, if not all, colorectal cancers arise. The evidence supporting this hypothesis has increased rapidly in recent years and the purpose of this article is to review this evidence critically and highlight its clinical signi®cance.Methods: Medline searches were used to identify recent key articles relating to the adenoma±carcinoma sequence. Further pertinent articles were obtained by manual scanning of the reference lists of identi®ed papers.Results: The evidence supporting the adenoma±carcinoma sequence can be classi®ed as epidemiological, clinicopathological and genetic. The most recent and largest body of data relates to molecular genetic events and their cellular effects; however, many other approaches, such as cytogenetics, molecular cytogenetics and cytometry, have also yielded valuable information.Conclusion: Recent work continues to support the adenoma±carcinoma sequence, but there is a paucity of data on the interrelationship between different genetic mutations and on the relationship between molecular and other types of genetic abnormalities. The clinical utility of the observations described has yet to be fully realized and global genetic analysis of colorectal tumours may prove to be central in rational adenoma management. IntroductionColorectal cancer is the second leading cause of cancerrelated death in the Western world; in the UK there are currently around 30 000 new cases per annum and 17 000 related deaths 1 . In recent years our understanding of the cellular and molecular events underlying the development of colorectal cancer has improved immeasurably but, despite this and advances in surgery, radiotherapy and chemotherapy, the average 5-year survival rate remains around 40 per cent 1 . One of the most important fundamental concepts in colorectal cancer to emerge in recent years has been the adenoma±carcinoma sequence, a term that describes the stepwise progression from normal to dysplastic epithelium to carcinoma associated with the accumulation of multiple clonally selected genetic alterations. This concept not only provides an excellent model to study the genesis of invasive cancer, but also affords a means of preventing colorectal cancer by endoscopic removal of precursor lesions.The appropriate management of individuals with precursor adenomatous polyps (adenomas) is of the utmost importance. It is known that after removal of such polyps 30±35 per cent of patients will have further adenomas detected at 3±4 years 2±4 and this has led to a policy of endoscopic surveillance for all adenoma-bearers 5 . However, given that approximately 40 per cent of the Western population will develop adenomas 6±8 and only 3 per cent will go on to suffer from colorectal cancer, it is clear that only a small proportion of adenomas progress to malignancy. Unfortunately, there are no reliable criteria available that can predict adenoma progression or recurrence, and the important questions of which individuals...

Flavoprotein reductases play a key role in electron transfer in many physiological processes. We have isolated a cDNA with strong sequence similarities to cytochrome P-450 reductase and nitric-oxide synthase. The cDNA encodes a protein of 597 amino acid residues with a predicted molecular mass of 67 kDa. Northern blot analysis identified a predicted transcript of 3.0 kilobase pairs as well as a larger transcript at 6.0 kilobase pairs, and the gene was mapped to chromosome 9q34.3 by fluorescence in situ hybridization analysis. The amino acid sequence of the protein contained distinct FMN-, FAD-, and NADPH-binding domains, and in order to establish whether the protein contained these cofactors, the coding sequence was expressed in insect cells and purified. Recombinant protein bound FMN, FAD, and NADPH cofactors and exhibited a UV-visible spectrum with absorbance maxima at 380, 460, and 626 nm. The purified enzyme reduced cytochrome c, with apparent K m and k cat values of 21 M and 1.3 s ؊1 , respectively, and metabolized the one-electron acceptors doxorubicin, menadione, and potassium ferricyanide. Immunoblot analysis of fractionated MCF7 cells with antibodies to recombinant NR1 showed that the enzyme is cytoplasmic and highly expressed in a panel of human cancer cell lines, thus indicating that this novel reductase may play a role in the metabolic activation of bioreductive anticancer drugs and other chemicals activated by one-electron reduction.Flavin-containing enzymes catalyze a broad spectrum of biochemical reactions ranging from oxidase, dehydrogenase, and mono-oxygenase reactions. Most flavoproteins contain either FMN or FAD as prosthetic groups; however, a small number of enzymes contain both cofactors. In mammalian systems, NADPH cytochrome P-450 oxidoreductase (cytochrome P-450 reductase) was the first such enzyme isolated (1, 2), followed by several other dual flavin enzymes including nitric-oxide synthases (NOS) 1 in higher organisms (3, 4), and CYP102 (5) and sulfite reductase (6) in bacteria. More recently, the cDNA sequence encoding a putative FMN-and FAD-binding enzyme, methionine synthase reductase, has been described (7). Cytochrome P-450 reductase, the most extensively characterized of these enzymes (8 -10), is found in the endoplasmic reticulum of most eukaryote cells and is an integral component of the monooxygenase system transferring electrons from NADPH to cytochromes P-450 via FMN and FAD co-factors. Cytochrome P-450 reductase may also donate electrons to heme oxygenase (11), cytochrome b 5 (12), and the fatty acid elongation system (13), and can reduce cytochrome c (14). Both the crystal and NMR structure of the FMN domain of human cytochrome P-450 reductase (15, 16) and the crystal structure of the NH 2 -terminally truncated form of the rat enzyme (17) have been resolved, providing high resolution structural information on this enzyme class. The amino-terminal region of cytochrome P-450 reductase bears striking amino acid homology with FMN-containing flavodoxins, while the carboxyl-term...

CCND1 encodes for the cyclin D1 protein involved in G1/S cell cycle transition. In breast cancer the mechanism of CCND1 amplification, relationship between cyclin D1 protein expression and the key clinical markers estrogen receptor (ER) and HER2 requires elucidation. Tissue microarrays of primary invasive breast cancer from 93 women were evaluated for CCND1 amplification by fluorescent in-situ hybridization and cyclin D1 protein overexpression by immunohistochemistry. CCND1 amplification was identified in 27/93 (30%) cancers and 59/93 (63%) cancers had overexpression of cyclin D1. CCND1 amplification was significantly associated with cyclin D1 protein overexpression (p < 0.001; Fisher's exact test) and both CCND1 amplification and cyclin D1 protein expression with oestrogen receptor (ER) expression (p 5 0.003 and p < 0.001; Fishers exact test). Neither CCND1 amplification nor cyclinD1 expression was associated with tumor size, pathological node status or HER2 amplification, but high CCND1 amplification (Copy Number Gain (CNG) ! 8) was associated with high tumor grade (p 5 0.005; chi square 7.915, 2 df) and worse prognosis by Nottingham Prognostic Index (p 5 0.001; 2 sample t-test). High CCND1 amplification (CNG ! 8) may identify a subset of patients with poor prognosis ER-positive breast cancers who should be considered for additional therapy.

Although the occurrence of both chromosomal aberrations and specific gene mutations in colorectal tumorigenesis is firmly established, the relationship between these different forms of genetic abnormality remains poorly understood. We have previously demonstrated, in colorectal adenocarcinomas, that mutations of APC, KRAS, and TP53 are each specifically associated with certain chromosomal aberrations. Using comparative genomic hybridization and mutational analysis of APC, KRAS, and TP53 to evaluate 78 colorectal adenomas, we have shown that several of the significant relationships between gene mutations and chromosomal abnormalities reported in colorectal adenocarcinomas also exist at the adenomatous stage. KRAS mutation correlated with 12p gain (P < 0.001) and TP53 mutation with both 20q gain and 18q loss (P = 0.03 for both). In addition, we have identified two chromosomal aberrations, gain of 13q and loss of 11q, that correlate with the presence of synchronous adenomas (P = 0.049 and P = 0.03, respectively) and several chromosomal changes (20p+, 20q+, 17p-, and 18q-) that are related to the onset of high-grade dysplasia. These data strengthen our previous contention that the co-occurrence of specific gene mutations and chromosomal changes is not random and significant relationships do exist. Our findings also raise the possibility that certain chromosomal aberrations may act as important clinical biomarkers.

The proband (fig 1) was the third child of healthy, unrelated parents. Pregnancy and delivery were uneventful, gestation was about 37 weeks, birth weight was 2.15 kg, and head circumference was 34 cm. The head was dolichocephalic with frontal bossing and the ears were low set and poorly lobulated. Hypertelorism, macrostomia, and micrognathia were observed, and the bridge of the nose was broad and depressed and the tip turned down-

Summary A proportion of cytogenetic abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) may escape detection by high‐resolution genomic technologies, but can be identified by conventional cytogenetic and molecular analysis. Here, we report the detection of a reciprocal translocation t(7;21)(p22;q22) in the marrow of two adults with MDS and AML, using conventional cytogenetic analysis and fluorescence‐in situ‐hybridization (FISH). Reverse‐transcription polymerase chain reaction (RT‐PCR) and sequence analysis identified a fusion between RUNX1 and the gene encoding ubiquitin specific peptidase‐42 (USP42), with splice‐variants and variable break‐points within RUNX1. Combined cytomorphology and FISH studies in MDS marrow revealed abnormal RUNX1 signals within megakaryocytes, suggesting that the acquisition of t(7;21)(p22;q22) does not confer complete differentiation arrest and may represent an early genetic event in leukaemogenesis. Single nucleotide polymorphism‐arrays failed to detect additional sub‐microscopic genomic changes predisposing to or associated with t(7;21). Molecular analysis of 100 MDS and AML marrow specimens by RT‐PCR did not reveal new cases with the RUNX1‐USP42 fusion. Thus, our studies have identified t(7;21)(p22;q22) as a rare but recurrent abnormality in MDS/AML, with the existence of alternative spliced forms of the RUNX1‐USP42 transcript in different patients. Further studies are required to identify the potential contribution of these splice‐variants to disease heterogeneity.

The incidence of adenocarcinoma arising at the esophagogastric junction (EGJ) is increasing at a rate greater than that for any other form of solid malignancy. Commensurate with this, the incidence of histologically similar tumors arising in the gastric body and antral mucosa is declining. The increased incidence of the proximal group of tumors may reflect, in part, the higher prevalence of Barrett esophagus. These epidemiological features suggest that histologically similar tumors arising at the EGJ and from the distal stomach are different, which may be reflected in the genetic abnormalities that characterize the two groups of tumors. The purpose of this study was to screen genomic DNA from adenocarcinomas of the esophagus and stomach for regions of chromosomal imbalance, using comparative genomic hybridization to determine whether tumors at the EGJ (junctional tumors) have a different profile compared with tumors of the distal stomach. Tumor samples were derived from a series of 48 gastroesophageal adenocarcinomas (20 junctional and 28 distal) that were acquired prospectively from patients undergoing esophagogastrectomy. These tumors are characterized by several regions of chromosomal imbalance with no obvious correlation between most regions of abnormal copy number and tumor type. However, our study shows for the first time cytogenetic abnormalities (5p+ and 18q-) that identify statistically significant differences (P < 0.02 and < 0.05, respectively) between junctional and distal gastric tumors. These differences are gain of 5p (55% [11/20] of junctional tumors vs. 21% [6/28] of distal gastric tumors) and loss of 18q (25% [5/20] cases of junctional tumors vs. 4% [1/28] of distal tumors) segregating with tumors of the EGJ. These abnormalities may distinguish distinct tumor subtypes that are recognized in epidemiological and clinical studies but that are otherwise histologically identical.

Background:Multiparameter flow cytometry is a robust and reliable method for determining tumour DNA content applicable to formalin-fixed paraffin-embedded (FFPE) tissue. This study examined the clinical and pathological associations of DNA content in primary breast cancer using an improved multiparametric technique.Methods:The FFPE tissue from 201 primary breast cancers was examined and the cancers categorised according to their DNA content using multiparametric flow cytometry incorporating differential labelling of stromal and tumour cell populations. Mathematical modelling software (ModFit 3.2.1) was used to calculate the DNA index (DI) and percentage S-phase fraction (SPF%) for each tumour. Independent associations with clinical and pathological parameters were sought using backward stepwise Binary Logistic Regression (BLR) and Cox's Regression (CR) analysis.Results:Tumours were grouped into four categories based on the DI of the tumour cell population. Low DI tumours (DI=0.76–1.14) associated with progesterone receptor-positive status (P=0.012, BLR), intermediate DI (DI=1.18–1.79) associated with p53 mutant tumours (P=0.001, BLR), high DI (DI⩾1.80) tumours with human epidermal growth factor receptor 2 (HER2)-positive status (P=0.004, BLR) and ‘multiploid tumours' (two or more tumour DNA peaks) did not show any significant associations. Tumours with high SPF% (⩾10%) independently associated with poor overall survival (P=0.027, CR).Conclusion:Multiparametric flow analysis of FFPE tissue can accurately assess tumour DNA content. Tumour sub-populations associated with biomarkers of prognosis or likely response to therapy. The alterations in DNA content present the potential for greater understanding of the mechanisms underlying clinically significant biomarker changes in primary breast cancer.