Reclassification of tumour origin in resected periampullary adenocarcinomas reveals underestimation of distal bile duct cancer.

BACKGROUND: Primary adenocarcinomas removed by pancreatoduodenectomy originate from the duodenum (DC), ampulla (AC), distal bile duct (DBC), or pancreas (PC). Pathobiology, staging, survival, and adjuvant chemotherapy vary among these cancers. The proximity of the structures of possible origin renders it difficult to obtain a correct diagnosis, which might lead to inconsistencies in reported data and inappropriate adjuvant treatment. METHODS: Records of 207 patients undergoing pancreatoduodenectomy (1998-2009) for periampullary adenocarcinoma were reviewed. Routine histopathology reports of tumour origin performed by multiple pathologists were independently re-evaluated based on predetermined criteria by two experienced pancreatic pathologists. RESULTS: Slide review changed the diagnosis in 55 (27%) patients. After reclassification, final distribution was 29 (14%) DC, 52 (25%) AC, 57 (28%) DBC, and 69 (33%) PC. The diagnosis was revised in 4 (14%) DC, 7 (17%) AC, 30 (53%) DBC and 14 (19%) PC. The underestimation of DBC during routine histopathology was caused by misinterpretation of DBC either PC or AC. Misclassification of PC was mainly due to erroneous diagnosis of AC. Reassignment of tumour origin caused no significant changes in survival within cancer type, but resulted in a significant difference in survival between DBC and PC (p = 0.004). CONCLUSION: Specialist slide review resulted in reassignment of tumour origin in 27% of periampullary adenocarcinomas. Distal bile duct cancer was found to be most frequently misdiagnosed (53%). Correct diagnosis of tumour origin is crucial for data quality, appropriate adjuvant therapy, and patient inclusion in clinical trials.

Reduced level of the spindle checkpoint protein BUB1B is associated with aneuploidy in colorectal cancers.

OBJECTIVES: The majority of solid human malignancies demonstrate DNA aneuploidy as a consequence of chromosomal instability. We wanted to investigate whether Aurora A, Aurora B, BUB1B and Mad2 were associated with the development of aneuploidy in colorectal adenocarcinomas as suggested by several in vitro studies, and if their protein levels were related to alterations at the corresponding chromosomal loci. MATERIALS AND METHODS: Expression levels of these spindle proteins were investigated by immunohistochemistry using tissue micro-arrays in a series of DNA aneuploid and diploid colorectal adenocarcinomas previously examined for genomic aberrations by comparative genomic hybridization. RESULTS: All proteins were overexpressed in malignant tissues compared to controls (P < 0.001 for all). BUB1B level was significantly reduced in aneuploid compared to diploid cancers (P = 0.001), whereas expression of the other proteins was not associated with DNA ploidy status. High levels of Aurora A (P = 0.049) and low levels of Aurora B (P = 0.031) were associated with poor prognosis, but no associations were revealed between protein expression and genomic aberration. CONCLUSIONS: A significant reduction of BUB1B level was detected in aneuploid compared to diploid colorectal cancers, consistent with earlier studies showing that loss of spindle checkpoint function may be involved in development of DNA aneuploidy. Our data also show that spindle proteins are overexpressed in colorectal cancers, and that expression of the Aurora kinases is associated with prognosis in colorectal cancer.

Spindle proteins Aurora A and BUB1B, but not Mad2, are aberrantly expressed in dysplastic mucosa of patients with longstanding ulcerative colitis.

BACKGROUND: Long term ulcerative colitis (UC) increases the risk of colorectal cancer (CRC). DNA aneuploidy is a common feature of both dysplastic and non-dysplastic colonic epithelia from patients with longstanding UC, and is regarded as an early sign of possible malignant transformation. The spindle proteins Aurora A, BUB1B and Mad2 have been implicated as contributors to aneuploidy and carcinogenesis. AIMS: To investigate the role of these spindle proteins in relation to DNA aneuploidy and during the progressive morphological changes in ulcerative colitis associated colorectal cancer (UCCRC). METHODS: Tissue microarrays were made from 31 colectomy specimens from patients with longstanding UC. Expression of Aurora A, BUB1B and Mad2 was investigated by immunohistochemistry and their relation to ploidy status, mucosal morphology and Ki67 levels was explored. RESULTS: Expression of Aurora A and BUB1B was significantly associated with the progressive morphological changes of UCCRC. In the progression from non-dysplastic to dysplastic mucosa, Aurora A expression decreased while BUB1B expression increased. There was an increasing incidence of aneuploidy with progression towards cancer; expression of all spindle proteins was associated with the level of Ki67 but not with aneuploidy. CONCLUSION: Due to the significant differences in Aurora A and BUB1B expression in dysplastic compared non-dysplastic mucosa, these proteins may serve as putative biological markers for the progressive morphological changes in UC associated carcinogenesis. The close relationship to Ki67 levels reflect that spindle proteins are expressed in tissues with a high proliferative rate; a role for these proteins in the development of aneuploidy was not found.

Genetic and protein markers related to in situ growth and multiplicity in small sporadic colorectal adenomas.

BACKGROUND: Some early genetic events in the development of colorectal adenomas are known, but their relationship to in vivo growth characteristics is uncertain. This study compared in situ size changes and other clinicopathological variables with selected genetic and protein markers. METHODS: 56 adenomas (< or = 10 mm) from 39 patients were analysed for APC, CTNNB1 and K-ras mutations, allelic imbalance on 1p and 18q, microsatellite instability and immunohistochemical expression of HLA-DR, BAX, BCL-2 and Ki-67. For 42 of the adenomas, in situ growth was measured over 3 years. The total number of polyps in each patient was recorded. RESULTS: K-ras was mutated in 8/56 adenomas. None of the regressing adenomas revealed such mutations, compared to 20% in those that maintained or increased their size. Multivariate linear regression analysis showed that tumour growth was higher in females compared to males, and was even higher in the presence of a K-ras mutation. APC mutations were found in 37/56 adenomas. CTNNB1 mutations were found in 2/19 adenomas without APC mutation. Deletions of 1p were found in 12/56 adenomas and, seemingly, most frequent in patients with few tumours. The most frequently expressed protein was BAX (33/41), but neither this nor the other proteins showed associations with an in situ growth pattern. CONCLUSION: The multivariate linear regression model showed that patient gender and the presence of K-ras mutation had significant effects on tumour growth. The lack of the proliferative stimulus resulting from a K-ras mutation may contribute to the process of adenoma regression.

The cyclosporin PSC 833 increases survival and delays engraftment of human multidrug-resistant leukemia cells in xenotransplanted NOD-SCID mice.

Circumvention of chemoresistance in cancer may involve several modulator drugs with high affinity for the multidrug transporter P-glycoprotein (Pgp), which is expressed in a number of multi-resistant malignancies. Pgp acts as a membrane efflux pump with broad substrate specificity including antineoplastic drugs and endogenous substances such as certain cytokines and sphingolipids. Therefore, the consequence of Pgp blockade could be far more complex than intracellular drug retention. In the present study exposure of the Pgp inhibitor, PSC 833 (1200 ng/ml), to Pgp expressing KG1a/200 human leukemia cells provoked cell cycle arrest and apoptosis in vitro. This finding was put to test in vivo using a xenotransplant model of KG1a/200 human cells intravenously inoculated into non-obese diabetic severe combined immunodeficient (NOD-SCID) mice. The animals were randomly allocated to receive treatment with PSC 833 (n = 32) or placebo (n = 24). PSC 833 (30 mg/kg) was subcutaneously injected six or 12 times separated by 48-96 h. The overall mean whole blood concentration of PSC 833 was 1191 +/- 60 ng/ml (s.e.m.) at 20 h after administration. Tumor engraftment was significantly reduced in the treatment group (P = 0.037), which also had prolonged survival compared to control animals (P = 0.0016). This is the first study that demonstrates antileukemic effects of a Pgp inhibitor as single agent therapy in vivo, and the present data raise the possibility of alternative exploitation of modulators in cancer chemotherapy.

Patients with small duct primary sclerosing cholangitis have a favourable long term prognosis.

BACKGROUND: Patients with cholestatic liver function tests and histological features of primary sclerosing cholangitis (PSC) but a normal cholangiogram are considered to have small duct PSC. The natural history of this condition is unknown. METHODS: Thirty three patients with small duct PSC were identified among patients admitted for diagnostic workup of cholestatic liver function tests in one centre in the UK (Oxford) and one centre in Norway (Oslo). A total of 260 patients with large duct PSC were compared, and prognosis in terms of death, cholangiocarcinoma, biochemical features, histological features, and symptoms analysed. RESULTS: Mean age at diagnosis was 38 years and 39 years in small duct and large duct PSC, respectively. Mean follow up was 106 months in small duct and 105 months in large duct patients. Four patients originally considered to have small duct developed large duct PSC. Two of these underwent liver transplantation during follow up. Of the remainder who did not develop large duct PSC, two patients died during follow up: one of liver failure and the other of cardiac death unrelated to her liver disease. A total of 122 (47%) large duct patients either required liver transplantation (34 patients) or died (88 patients). Small duct patients had a significantly better survival compared with large duct patients. Among small duct patients, none developed cholangiocarcinoma compared with 28 of 260 (11%) large duct patients. CONCLUSIONS: Patients with small duct PSC seem to have a good prognosis in terms of survival and development of cholangiocarcinoma. Small duct PSC progresses to large duct PSC in a small proportion of patients.

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.

Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis.

We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis.

Biliary dysplasia as a marker of cholangiocarcinoma in primary sclerosing cholangitis.

BACKGROUND/AIMS: Indentification of biliary dysplasia in a primary sclerosing cholangitis (PSC) liver biopsy may indicate developing cholangiocarcinoma. The objectives were to determine whether biliary dysplasia can be recognised reproducibly in PSC and to compare the frequency in cases with and without cholangiocarcinoma. METHODS: Liver biopsies from 26 PSC cases with concurrent or subsequent cholangiocarcinoma (within 2 years) were assessed for biliary dysplasia independently by three liver pathologists. This was done in two stages: initially, without agreement on criteria, and subsequently after such agreement. Liver biopsies from 60 PSC cases without cholangio-carcinoma were also assessed. RESULTS: Reproducibility for biliary dysplasia without prior agreement on criteria was only marginally better than random (kappa=0.129). In contrast, after prior agreement on criteria, reproducibility was moderate (kappa=0.44). Biliary dysplasia was agreed to be present by all three pathologists in 23% and 19% of biopsies in the first and second round, respectively, from patients with cholangiocarcinoma, but in none of the patients without cholangiocarcinoma. CONCLUSION: Criteria for biliary dysplasia can be agreed and the entity recognised in liver biopsies. The strong association of biliary dysplasia with cholangiocarcinoma in PSC suggests use of dysplasia as a marker for current or developing malignancy.

Prognostic significance of recurrent chromosomal aberrations detected by comparative genomic hybridization in sporadic colorectal cancer.

Colorectal carcinomas are characterized by frequent recurrent gains and losses of chromosomal material, especially gains of chromosome arms 20q and 13q, and losses of chromosome arms 18q and 4q. These may be important in the development and progression of colorectal carcinomas. Chromosomal aberrations detected by comparative genomic hybridization in 67 sporadic colorectal carcinomas were examined for their possible associations with patient survival. Dukes' stage, tumor DNA ploidy status, and TP53 genotype/phenotype were also examined for the same. Patients with losses of chromosomal arms 1p, 4q, 8p, 14q, or 18q or gain of chromosomal arm 20q had significantly shorter survival times than those without these aberrations (univariate relative risk 3.45, 2.71, 3.32, 3.26, 3.32, 3.91, respectively), as did patients with more than six chromosomal aberrations per tumor than those with fewer than six aberrations (univariate relative risk 3.26, P = 0.013). DNA aneuploidy and Dukes' stage C + D resulted in poor patient survival (univariate relative risk 3.58, 3.39, respectively). Dukes' stage C + D, 1p loss and 8p loss emerged as the only independent prognostic parameters (relative risk 3.22, 2.53, 2.45, respectively) when entered into multivariate survival analysis together with other significant parameters from univariate survival analysis. Loss of chromosome arm 1p, 4q, 8p, 14q, or 18q or gain of chromosome arm 20q thus results in shortened survival times in colorectal cancer patients. 1p loss and 8p loss were shown to be independent predictors of poor prognosis.

A strategy combining flow sorting and comparative genomic hybridization for studying genetic aberrations at different stages of colorectal tumorigenesis in ulcerative colitis.

BACKGROUND: DNA aneuploidy has been shown to increase the risk of developing dysplasia in ulcerative colitis (UC) and is related to tumorigenesis in the colorectum. Therefore, it is of particular interest to study genetic aberrations behind DNA aneuploidization during colorectal carcinogenesis. We wanted to elucidate further the relationship between mucosal morphology and DNA aberrations in UC. METHODS: DNA flow cytometry was applied to multiple lesions including regenerative, dysplastic, and carcinomatous mucosa from the colectomy specimen of a male patient with long-standing UC. The lesions harbored multiple DNA aneuploid stemlines that were subjected to flow sorting. We analyzed gene alterations by degenerate oligonucleotide primer (DOP; universal primers) polymerase chain reaction (PCR)-based comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) in diploid and aneuploid sorted cells. RESULTS: DOP-PCR-based CGH shows gains and losses that can be verified by FISH. We show that with this approach one can study genetic evolution of distinct DNA diploid and aberrant subpopulations through defined stages of colorectal tumorigenesis. This includes getting information related to tumor heterogeneity that cannot be obtained by CGH with DNA extracted from nonsorted cell populations. Genetic imbalance was also detected in diploid nondysplastic flow-sorted mucosal cells from the same bowel. CONCLUSIONS: Similar gains and losses were found in aneuploid dysplasias and carcinomas at widely separated locations in the same bowel, indicating a common selection pressure in different areas of the same bowel. The common aberrations may be of importance for progression from dysplasia to carcinoma.

p53 accumulation confers prognostic information in resectable adenocarcinomas with ductal but not with intestinal differentiation in the pancreatic head.

The aim of the study was to examine the relation between p53 protein accumulation, clinicopathological variables and prognosis in resectable adenocarcinomas of the pancreatic head. The clinical records and tissue specimens of 82 consecutive patients resected for adenocarcinomas located in the head of the pancreas were reviewed retrospectively. Formalin-fixed and paraffin-embedded specimens from each tumour were stained with the monoclonal antibody DO7, and the nuclear p53 positivity within each tumour was assessed. Histopathological reclassification showed that 60 tumours exhibited ductal differentiation and 22 tumours intestinal differentiation. Twenty-five percent (15/60) of the ductal tumours and 50% (11/22) of the intestinal tumours were positive for p53 accumulation. p53 immunoreactivity was significantly correlated to a worse prognosis in the tumours of ductal differentiation, with median survival 0.76 years for p53 positive and 1.44 years for p53 negative patients. The p53 positivity of tumours with intestinal differentiation showed no such correlation. No correlation was found between p53 accumulation and other known prognostic factors in either the ductal or the intestinal type of tumours. Our results indicate that the tumour biology of ductal adenocarcinomas differs significantly from that of adenocarcinomas of the intestinal type located in the pancreatic head, and that p53 accumulation confers a worse prognosis only of ductal tumours. Subclassification of these tumours based on type of differentiation is therefore suggested since periampullary tumours include ductally as well as intestinally differentiated adenocarcinomas.

[Flow cytometry--use in cell biology and benefits in clinical medicine]. / Vaeskestrømscytometri--anvendelse i cellebiologi og nytte i klinisk medisin.

Flow cytometry is an automated method for measuring features of single cells in suspension. Many parameters may be measured simultaneously at high speed, objectively and with good statistics. The method has been increasingly used in the context of medical diagnosis and prognosis over the last ten years. Clinical-chemical departments are major users of flow cytometry for differential cell counts of leukocyte populations in blood by light scatter measurements. For immunophenotyping of acute leukaemias, flow cytometry is used routinely in all university hospitals in Norway. The method is also used to classify chronic lymphoproliferative disease, and for quantification of CD34-positive stem cells by high-dose chemotherapy with autologous stem cell support. Flow cytometry is routinely used for classifying non-malignant haematological disease like congenital and acquired immunodeficiencies. In diagnostic pathology, DNA flow cytometry is used for analysis of DNA ploidy and S-phase fractions in malignant tumours. DNA aneuploidy is a strong prognostic factor in some childhood tumours and in gynaecological malignant tumours, and flow cytometry is used in tumour biology for measurements of growth and apoptotic cell death. Furthermore, the method is an important research tool in many areas of biomedical research for detection of structural and functional cellular features.

Cyclosporin A inhibits cardiac hypertrophy and enhances cardiac dysfunction during postinfarction failure in rats.

Calcineurin has recently been implicated as a mediator in the signaling pathways that transform intracellular calcium signals to the phenotype of myocardial hypertrophy. The present study was conducted to examine the effects of cyclosporin A (CsA), an inhibitor of calcineurin, on myocardial hypertrophy and remodeling during congestive heart failure (CHF) in rats. After ligation of the left coronary artery, rats were randomized to treatment with CsA or vehicle for 14 days. Compared with vehicle, CsA substantially attenuated myocardial hypertrophy in the CHF rats as assessed by alterations in ventricular weight-to-tibial length ratios (P < 0.05). Myocardial gene expression of skeletal alpha-actin was also reduced in the failing left ventricle (LV) after treatment with CsA (P < 0. 05), although the mRNA levels were still substantially elevated relative to those of sham rats. CsA-induced inhibition of compensatory myocardial hypertrophy in the CHF rats led to increased dilatation of the LV cavity and reduced fractional shortening and peak positive and negative first derivatives of LV pressure (P < 0. 05). Plasma renin and endothelin-1 levels were increased in the CHF-CsA rats, providing humoral cues of aggravated cardiac function. Thus this study supports a crucial role of calcineurin-dependent pathways in the mechanisms of compensatory myocardial hypertrophy during CHF. In addition, our data indicate that inhibition of compensatory myocardial hypertrophy exerts detrimental effects on cardiac remodeling and function after myocardial infarction.

Cholangiocarcinoma in primary sclerosing cholangitis: K-ras mutations and Tp53 dysfunction are implicated in the neoplastic development.

BACKGROUND/AIM: Cholangiocarcinoma is a feared complication of primary sclerosing cholangitis (PSC). Neoplastic bile duct strictures may be difficult to differentiate cholangiographically from the non-neoplastic bile duct irregularities characteristic of this disorder, and the diagnosis of cholangiocarcinoma may be difficult to establish with certainty, even in tissue samples. Thus, new methods which can improve the diagnostic accuracy of cholangiocarcinoma in PSC are needed. METHODS: We investigated the occurrence of K-ras codon 12 and 13 mutations, p53 protein accumulation, and Ki-67 expression in tumor tissue from PSC patients (n=33) who had developed cholangiocarcinoma, using bile duct specimens exised at liver transplantation of PSC patients without cholangiocarcinoma (n=15) as controls RESULTS: K-ras mutations were present in 11 (33%) of the cholangiocarcinoma samples and significantly more frequent in females. Nine tumors carried a codon 12 mutation, and 2 had a codon 13 mutation. The most frequent substitutions in codon 12 were GGT-->GAT (n=5) and GGT-->TGT (n=3). None of the control bile ducts had K-ras mutations. p53 protein was accumulated in 10 (31%) of the tumors, as opposed to negative findings in all the control samples. Sixteen (48%) tumors revealed either K-ras mutation or p53 accumulation. Ki-67 positivity was significantly higher in cholangiocarcinomas than in the non-neoplastic bile ducts (median 29% vs 12%, respectively; p=0.011). CONCLUSION: We conclude that K-ras mutations and p53 dysfunction are implicated in tumorigenesis of cholangiocarcinomas arising in PSC patients and that these abnormalities together with increased Ki-67 index may indicate neoplastic progression of bile ducts in these patients.

Grzybowski's generalized eruptive keratocanthomas: a case report.

A 47-year old woman with Grzybowski's generalised eruptive keratoacanthomas is described. There was no history of skin disease in her family, except for an uncle's basal cell carcinoma. From 1995 she developed multiple lesions of various size, ranging from hundreds of small follicular lesions to large typical keratoacanthomas up to 5 cm in diameter, scleroderma-like facial skin and marked ectropion. Histological examination of small and large skin lesions was typical of keratoacanthoma, and no human papillomavirus was detected by polymerase chain reaction. Oral treatment with acitretin had no effect. Both cyclophosphamide and methotrexate therapy were refused by the patient despite the progressive course of the disease. Blepharoplastic surgery had some effect on eye symptoms. The etiology of this rare disease is unknown, but is probably related to some genetic defect.

Villous, hypermucinous mucosa in long standing ulcerative colitis shows high frequency of K-ras mutations.

BACKGROUND: K-ras mutation is one of the first genetic alterations in classical colorectal carcinogenesis. AIMS: To investigate the role of K-ras mutations in carcinogenesis, in long standing ulcerative colitis. METHODS: A total of 161 microdissected and 100 DNA samples from 13 patients were analysed for K-ras codons 12 and 13 mutations by means of a combination of enriched polymerase chain reaction amplification and temporal temperature gradient electrophoresis. RESULTS: K-ras mutations were found in 21/161 (13%) microdissected samples in 7/13 large bowels (16 and five in codons 12 and 13, respectively), and in 10/100 (10%) mucosal DNA samples (six and four, respectively). One of four patients with six adenocarcinomas had a K-ras mutation in a carcinoma, as well as one of two patients with large dysplasia associated lesion or mass (DALM). Eight of 13 (61%) areas with villous architecture and large, distended goblet cells, had a K-ras mutation, which was significantly more frequent than in low grade dysplasia (one of 23, 4%) but did not reach significance versus high grade dysplasia (four of 14, 28.5%). K-ras mutations were found in one of 20 (5%) flat lesions indefinite for dysplasia, two of 14 (14%) in non-villous, hypermucinous mucosa, and in one of 57 flat areas negative for dysplasia. CONCLUSION: The highest K-ras mutation frequency was found in villous, hypermucinous mucosa. We suggest that this entity should be investigated further as a potential risk lesion for cancer development. It may represent a pathway directly from non-classical dysplasia to cancer, not previously described.

The prognostic value of spontaneous apoptosis, Bax, Bcl-2, and p53 in oral squamous cell carcinoma of the tongue.

BACKGROUND: Bax, Bcl-2, and p53 proteins are involved in the regulation of apoptosis and have been reported to correlate with prognosis in several tumor types. METHODS: Bax, Bcl-2, p53, and the level of spontaneous apoptosis were evaluated in formalin fixed, paraffin embedded pretreatment specimens from 85 T1-4 squamous cell carcinomas (SCCs) of the tongue by immunohistochemical methods. The percentage of apoptotic cells labeled by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP labeling (TUNEL) method was expressed as an apoptotic index (AI). For Bax and Bcl-2 evaluation, the fraction of tumor cells stained and the staining intensities were given scores that were added together, resulting in a final score. p53 immunostaining was expressed as a percentage of positive cells. RESULTS: High AI was significantly associated with high Bax expression (P = 0.0122) and highly differentiated tumors (P = 0.0062). No correlation was found between AI and Bcl-2 expression. There was no correlation between p53 positivity and any of the other apoptosis-related parameters. Whereas low AI scores and low Bax expression correlated significantly with poor prognosis (P = 0.0053 and P = 0.0012, respectively), a low Bcl-2 expression was associated with a favorable clinical outcome (P = 0.0262). Patients with a high Bcl-2/Bax expression ratio had a significantly poorer prognosis than those with a low ratio (P < 0.0001). Multivariate analysis revealed that Bax expression, the Bcl-2/Bax expression ratio, and the T and N classifications were significantly independent prognostic variables. The Bcl-2/Bax expression ratio was the strongest independent prognostic parameter. CONCLUSIONS: AI, individual Bax and Bcl-2 expression, and particularly the Bcl-2/Bax expression ratio have prognostic value in SCC of the tongue.

Prognostic value of Le(y) and H antigens in oral tongue carcinomas.

OBJECTIVE: To investigate the prognostic significance of the carbohydrate epitopes H and Le(y) and their relationship with proliferation and apoptosis. STUDY DESIGN: Eighty randomly selected patients with T1-T4 oral tongue squamous cell carcinoma (SCC) were studied. Serial sections were cut from diagnostic, formalin-fixed, paraffin-embedded specimens. METHODS: Sections were stained immunohistochemically for H antigen and Le(y). RESULTS: Expression of H antigen was associated positively with Le(y) expression (P = .0001). Expressions of H antigen or Le(y) correlated with the proliferative markers Ki67 (P = .0442 and P = .0003, respectively) and pAgNOR > 1 (P = .0674 and P = .0047, respectively), but not with apoptotic markers such as Bax expression or the apoptotic index (AI). Tumors that expressed H antigen and high levels of Le(y) (> 50%) had a poor prognosis (P = .0006 and P = .0056, respectively). Combinations of expression of H antigen and Le(y), and either proliferative or apoptotic markers revealed an enhanced prognostic potential (P < .0001). The combination of pAgNOR score greater than 1 and H-antigen expression appeared to be the best combination to predict good prognosis. CONCLUSION: The expression of H antigen and Le(y), especially their combination with proliferative or apoptotic markers, has prognostic value in tongue SCC.

Chromosomal gains and losses in primary colorectal carcinomas detected by CGH and their associations with tumour DNA ploidy, genotypes and phenotypes.

Comparative genomic hybridization (CGH) is used to detect amplified and/or deleted chromosomal regions in tumours by mapping their locations on normal metaphase chromosomes. Forty-five sporadic colorectal carcinomas were screened for chromosomal aberrations using direct CGH. The median number of chromosomal aberrations per tumour was 7.0 (range 0-19). Gains of 20q (67%) and losses of 18q (49%) were the most frequent aberrations. Other recurrent gains of 5p, 6p, 7, 8q, 13q, 17q, 19, X and losses of 1p, 3p, 4, 5q. 6q, 8p, 9p, 10, 15q, 17p were found in > 10% of colorectal tumours. High-level gains (ratio > 1.5) were seen only on 8q, 13q, 20 and X, and only in DNA aneuploid tumours. DNA aneuploid tumours had significantly more chromosomal aberrations (median number per tumour of 9.0) compared to diploid tumours (median of 1.0) (P < 0.0001). The median numbers of aberrations seen in DNA hyperdiploid and highly aneuploid tumours were not significantly different (8.5 and 11.0 respectively; P = 0.58). Four tumours had no detectable chromosomal aberrations and these were DNA diploid. A higher percentage of tumours from male patients showed Xq gain and 18q loss compared to tumours from female patients (P = 0.05 and 0.01 respectively). High tumour S phase fractions were associated with gain of 20q13 (P = 0.03), and low tumour apoptotic indices were associated with loss of 4q (P = 0.05). Tumours with TP53 mutations had more aberrations (median of 9.0 per tumour) compared to those without (median of 2.0) (P = 0.002), and gain of 8q23-24 and loss of 18qcen-21 were significantly associated with TP53 mutations (P = 0.04 and 0.02 respectively). Dukes' C/D stage tumours tended to have a higher number of aberrations per tumour (median of 10.0) compared to Dukes' B tumours (median of 3.0) (P = 0.06). The low number of aberrations observed in DNA diploid tumours compared to aneuploid tumours suggests that genomic instability and possible growth advantages in diploid tumours do not result from acquisition of gross chromosomal aberrations but rather from selection for other types of mutations. Our study is consistent with the idea that these two groups of tumours evolve along separate genetic pathways and that gross genomic instability is associated with TP53 gene aberrations.