Diagnostic work-up of carcinoma of unknown primary: from immunohistochemistry to molecular profiling.

Carcinoma of unknown primary (CUP) remains a common and challenging clinical problem. The aim of diagnostic work-up in CUP is to classify as specifically as possible the cancer affecting the patient, according to the broad tumour type, subtype and, where possible, site of origin. This classification currently best predicts patient outcome and guides optimal treatment. a stepwise approach to diagnostic work-up is described. although pathology is based on morphology, the assessment of tissue-specific genes through immunohistochemistry (IHC) substantially helps tumour classification at each diagnostic step. For IHC in CUP, recent improvements include more standardised approaches and marker panels plus new markers. Tissue-specific genes are also being used in CUP work-up through molecular profiling. Large-scale profiles of hundreds of tumours of different types have been generated, compared and used to generate diagnostic algorithms. Commercial tests for CUP classification have been developed at the mRNa and microRNA and (miRNA) levels and validated in metastatic tumours and CUPs. While currently optimal pathology and IHC remain the 'gold standard' for CUP diagnostic work-up, and full clinical correlation is vital, the molecular tests appear to perform well: in the main diagnostic challenge of undifferentiated or poorly differentiated tumours, molecular profiling performs as well as or better than IHC.

Loss of MSH2 protein expression is a risk factor in early stage cervical cancer.

BACKGROUND: Loss of mismatch repair (MMR) gene expression has been associated with fewer metastases and improved prognosis in various tumour types. AIMS: To evaluate the predictive and prognostic significance of loss of MMR protein MSH2 in early stage cervical cancer. METHODS: Specimens from 218 consecutive patients with early stage, surgically treated cervical cancer were analysed. Median age was 42 years (interquartile range 35-53). International Federation of Gynecology and Obstetrics (FIGO) stages were IB1 (57%), IB2 (25%) and IIA (18%). Histology was 70% squamous cell, 6% adenosquamous and 24% adenocarcinoma. Pelvic lymph node metastasis was present in 66 (30%) patients. Median follow-up was 5.2 years (interquartile range 2.5-7.9). Tissue microarrays (TMAs) were constructed containing three cores of paraffin-embedded tumour per case. MSH2 expression was assessed by immunohistochemistry on TMAs and full sections. RESULTS: In TMAs MSH2 expression could be analysed in 184/218 (84%) tumours. Loss of MSH2 was observed in 58/184 (32%) tumours, with a moderately strong concordance between TMAs and full sections (kappa = 0.47). In tumours with loss of MSH2, pelvic lymph node metastasis and cancer invasion beyond 10 mm were more frequent (48% vs 25%, and 59% vs 37%, respectively). However, loss of MSH2 expression was not related to recurrence or survival. CONCLUSION: TMAs are powerful tools for high throughput screening of biological markers for prognostic value in cervical cancer. Absence of MSH2 expression is associated with a high-risk profile in early stage cervical cancer, but does not predict lymph node status with sufficient accuracy to be used in the clinic.

Drug- not carrier-dependent haematological and biochemical changes in a repeated dose study of cyclosporine encapsulated polyester nano- and micro-particles: size does not matter.

Biodegradable nanoparticles are being considered more often as drug carriers to address pharmacokinetic/pharmacodynamic issues, yet nano-product safety has not been systematically proven. In this study, haematological, biochemical and histological parameters were examined on 28 day daily dosing of rats with nano- or micro-particle encapsulated cyclosporine (CsA) to confirm if any changes observed were drug or carrier dependent. CsA encapsulated poly(lactide-co-glycolide) [PLGA] nano- (nCsA) and micro-particles (mCsA) were prepared by emulsion techniques. CsA (15, 30, 45 mg/kg) were administered by oral gavage to Sprague Dawley (SD) rats over 28 days. Haematological and biochemical metrics were followed with tissue histology performed on sacrifice. Whether presented as nCsA or mCsA, 45 mg/kg dose caused significant loss of body weight and lowered food consumption compared to untreated control. Across the doses, both nCsA and mCsA produce significant decreases in lymphocyte numbers compared to controls, commensurate with the proprietary product, Neoral(®) 15. Dosing with nCsA showed higher serum drug levels than mCsA presumably owing to the smaller particle size facilitating absorption. The treatment had no noticeable effects on inflammatory/oxidative stress markers or antioxidant enzyme levels, except an increase in ceruloplasmin (CP) levels for high dose nCsA/mCsA group. Further, only subtle, sub-lethal changes were observed in histology of nCsA/mCsA treated rat organs. Blank (drug-free) particles did not induce changes in the parameters studied. Therefore, it is extremely important that the encapsulated drug in the nano-products is considered when safety of the overall product is assessed rather than relying on just the particle size. This study has addressed some concerns surrounding particulate drug delivery, demonstrating safe delivery of CsA whilst achieving augmented serum concentrations.

A tale of two sisters.

Hereditary haemochromatosis is the most common inherited disorder in white populations, whereas non-alcoholic steatohepatitis (NASH) is becoming the most common reason for referral for investigation of abnormal liver function tests (LFTs). This report describes two sisters, from similar environments, who were referred to the clinic after being found to be C282Y homozygotes and to have abnormal LFTs. One sister had developed features of haemochromatosis and the other had developed NASH. These cases illustrate the potential non-penetrance of HFE gene mutations and the need to investigate abnormal LFTs fully, even when there is a positive genetic test at the outset.

Non-alcoholic steatohepatitis: a common cause of progressive chronic liver injury?

AIM: To investigate the natural history of patients with non-alcoholic steatohepatitis by means of a prospective histological study. METHODS: One thousand five hundred and seventy one patients underwent liver biopsy at the Western Infirmary in Glasgow during the 10 year period 1985 to 1994. All biopsies were reported by a single pathologist: 62 were confirmed as having non-alcoholic steatohepatitis and prospective follow up was conducted in 1999. Repeat liver biopsy was carried out where appropriate to assess disease progression. RESULTS: Initial biopsy scores for the 62 patients (20 men; mean age at biopsy, 52 years) showed a mean of 1.85, 1.39, and 0.5 for necroinflammation, fibrosis, and iron stores, respectively. Forty six were traceable and invited for review, and 26 attended (six men; mean age at initial biopsy, 49.9 years) at a mean of 8.7 years after the initial liver biopsy. No patients had symptoms or signs of chronic liver disease. Four patients had normal liver function tests, one had cirrhosis; the remaining 21 were invited to have a repeat biopsy. Seven patients agreed, a mean 8.2 years after the initial biopsy, and repeat biopsy scores showed no significant difference over this time period, with mean scores of 1.71 (initial score, 2.14), 1.43 (initial score, 0.71), and 0.14 (initial score, 0) for necroinflammation, fibrosis, and iron stores, respectively. CONCLUSION: In this series of patients with non-alcoholic steatohepatitis, with a mean clinical follow up of 8.7 years, and a histological follow up of 8.2 years, there was no evidence of progressive chronic liver injury.

Hepatic granulomas: a 10 year single centre experience.

BACKGROUND: Epithelioid granulomas have been reported in 2-15% of unselected liver biopsies, with numerous underlying aetiologies described. However, all UK series were reported before identification of hepatitis C virus (HCV). AIM: To evaluate the current aetiologies of hepatic granulomas and to assess the prognosis for the "idiopathic" group, in which all investigations for a recognised cause were negative or normal. METHODS: A retrospective review of patient case notes between 1991 and 2001; all patients who had a liver biopsy at Glasgow Royal Infirmary revealing epithelioid granulomas had their case notes and liver biopsies reviewed and a standard proforma completed. RESULTS: Over the study period, 1662 liver biopsies were performed. Hepatic granulomas were found in 63. Of those identified, 47 were female, with a mean age of 42 years (range, 17-81). Underlying aetiologies were as follows: primary biliary cirrhosis (PBC; 23.8%), sarcoidosis (11.1%), idiopathic (11.1%), drug induced (9.5%), HCV (9.5%), PBC/autoimmune hepatitis (AIH) overlap (6.3%), Hodgkin lymphoma (6.3%), AIH (4.8%), tuberculosis (4.8%), resolving biliary obstruction (3.2%), and other single miscellaneous causes (9.5%). Of the seven patients with idiopathic hepatic granulomas, one was lost to follow up, one died of stroke, and the remaining five were well with no liver related morbidity at a mean follow up of 6.2 years. CONCLUSIONS: The aetiology of hepatic granulomas is broad ranging, with HCV an important cause in this population. Despite extensive investigations, a 10-15% of patients still had "idiopathic" hepatic granulomas. However, the prognosis for this last group appears to be excellent.

Factors influencing p53 expression in ovarian cancer as a biomarker of clinical outcome in multicentre studies.

The prognostic impact of p53 immunostaining in a large series of tumours from epithelial ovarian cancer patients in a two-centre study was analysed. The study population (n=476) comprised of a retrospective series of 188 patients (Dutch cohort) and a prospective series of 288 patients (Scottish cohort) enrolled in clinical trials. P53 expression was determined by immunohistochemistry on tissue microarrays. Association with progression-free survival (PFS) and overall survival (OS) was analysed by univariate and multivariate Cox regression analysis. Aberrant p53 overexpression was significantly associated with PFS in the Dutch and Scottish cohorts (P=0.001 and 0.038, respectively), but not with OS in univariate analysis. In multivariate analysis, when the two groups were combined and account taken of clinical factors and country of origin of the cohort, p53 expression was not an independent prognostic predictor of PFS or OS. In this well-powered study with minimal methodological variability, p53 immunostaining is not an independent prognostic marker of clinical outcome in epithelial ovarian cancer. The data demonstrate the importance of methodological standardisation, particularly defining patient characteristics and survival end-point data, if biomarker data from multicentre studies are to be combined.

Polymorphisms of the renin-angiotensin system and the severity of fibrosis in chronic hepatitis C virus infection.

SUMMARY: Patients with chronic hepatitis C virus (HCV) infection vary in their rates of fibrosis progression. The renin-angiotensin system (RAS) regulates fibrosis. Polymorphisms in the genes of the RAS may contribute to the outcome of renal and cardiovascular disease. We studied four RAS gene polymorphisms in 195 patients with chronic HCV infection. Patients were grouped by Ishak stage of fibrosis on liver biopsy: group 1 (fibrosis score 0 or 1; n = 97), group 2 (fibrosis score 2 or 3; n = 73) and group 3 (fibrosis score 4-6; n = 25). Polymorphisms of the angiotensinogen (AGT) gene (M235T and AT-6), the angiotensin I converting enzyme gene and the type 1 angiotensin II receptor gene were assayed. There was no difference in the distribution of these polymorphisms of the RAS between the fibrosis groups. There did not appear to be any increased prevalence of fibrosis if two or even three of the polymorphisms associated with increased RAS effect were present. On multivariate analysis factors significantly associated with fibrosis were necroinflammatory activity (P < 0.001) and age (P < 0.001). No association was identified between these four RAS polymorphisms and fibrosis in chronic HCV infection.

Predictive value of thymidylate synthase and dihydropyrimidine dehydrogenase protein expression on survival in adjuvantly treated stage III colon cancer patients.

BACKGROUND: The predictive value of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression on long-term survival by influencing 5-fluorouracil (5-FU) effect were determined in primary tumours and node metastases of stage III colon cancer patients treated adjuvantly with 5-FU regimens (n=391). The effect of TP 53 mutation status, which is thought to be functionally linked to TS inhibition, was also examined. PATIENTS AND METHODS: TS and DPD protein expression was determined by immunohistochemical analysis using tissue microarrays of these colon tumours. Two hundred and twenty tumours had already been screened in a previous study for TP 53 mutations. RESULTS: Low TS protein levels in primary stage III colon tumours appeared to be associated with mucinous histology and low DPD protein levels with young age at time of randomisation. Concordance between TS and DPD expression in primary and metastatic tumours was low. No associations were found between disease-free survival (DFS) and TS or DPD protein levels. When stratified by TP 53 mutation status DFS did not differ with TS expression. CONCLUSIONS: Expression of TS and DPD proteins is not predictive for survival in patients with stage III colon cancer treated adjuvantly with 5-FU regimens. TS protein levels did not alter the effect of TP 53 mutation status.

Hepatocellular carcinoma after long-term tamoxifen therapy.

We describe a case of hepatocellular carcinoma (HCC) after long term tamoxifen therapy in a 71-year-old woman. The patient was prescribed tamoxifen for 12 years following right mastectomy and axillary node clearance for breast carcinoma in 1985. In 1997, she complained of abdominal pain and fullness. An abdominal ultrasound scan showed lesions in the right lobe of liver which were thought to be metastases. However, a biopsy showed primary HCC. Studies in rats suggest that tamoxifen is involved in hepatic carcinogenesis but studies in humans have failed to show any increased risk. However, these studies followed up patients for less than five years. An increased risk of HCC may not become apparent until after a decade or more of tamoxifen therapy. In addition, HCC in tamoxifen treated patients may be under-reported since there may be reluctance to biopsy liver tumours which are assumed to be secondary carcinoma of the breast.

Improvement in cholestasis associated with total parenteral nutrition after treatment with an antibody against tumour necrosis factor alpha.

BACKGROUND: Many patients receiving long-term total parenteral nutrition (TPN) develop liver disease; cholestasis is common and may be severe. Antitumour necrosis factor alpha (TNFalpha) antibodies have recently been used in order to treat Crohn's disease, but their effect on cholestasis in humans has not been previously described. CASE REPORT: A 45-year-old woman had complicated Crohn's disease with multiple fistulae and only 1 m of residual small bowel. She had been receiving TPN for 2.5 years when she developed cholestasis which worsened despite adjustments to her TPN regimen. Infliximab, an anti-TNFalpha antibody, was given with the aim of treating an enterocutaneous fistula, but it also produced a marked biochemical and histological improvement in the TPN-related cholestasis. CONCLUSIONS: Anti-TNFalpha antibodies appeared in this case to improve TPN-related cholestasis. This implies that TNFalpha may play an important role in the development of this condition.

Frequency of early colorectal cancer in patients undergoing colonoscopy.

BACKGROUND: Early colorectal cancer is defined as carcinoma limited to the mucosa or submucosa. Up to 20 per cent of all colorectal cancers treated in some Japanese institutions are early cancers. These cancers are sometimes flat or depressed, and may be less than 1 cm in diameter. The aim of this study was to identify the frequency and morphology of early colonic cancers detected at colonoscopy by a surgeon aware of and looking for such lesions. METHODS: A review was made of all colonoscopies performed by or under the supervision of a single endoscopist between 1990 and 1998. Follow-up and outcome of all patients with early colorectal cancer was undertaken. RESULTS: Ninety-five invasive colorectal cancers were identified from 2198 colonoscopies. Eighteen were early colorectal cancers (T1). Macroscopically these were flat (nine tumours), villous (four) and pedunculated (five). Two patients had lymph node metastasis. The median size of flat cancers was 20 (range 9-30) mm. Median follow-up was 3 years. One patient had local recurrence, and another, whose early cancer was metachronous, died from metastatic cancer. CONCLUSION: This study identified early colonic cancer with similar frequency and morphology to that reported by the Japanese. Colonoscopy should be considered as the investigation of choice for patients with large bowel symptoms.

Co-amoxiclav jaundice: clinical and histological features and HLA class II association.

BACKGROUND AND AIMS: Jaundice associated with co-amoxiclav has been increasingly recognised. We aimed to characterise its clinical and histological features and to investigate linkage with human leucocyte antigen class II haplotypes. METHODS: We identified cases in the west of Scotland in the period 1991-1997 and performed polymerase chain reaction amplification and oligonucleotide probing on whole blood. RESULTS: Twenty two cases were identified (10 male, mean age 59.1 years). Jaundice occurred a median of 17 days after drug commencement, with a median peak bilirubin level of 225 micromol/l (range 84-598) and median duration of jaundice 69 days (range 29-150). Two patients had primary biliary cirrhosis and two other patients had persistently abnormal liver biochemistry on follow up. One death occurred in a frail elderly woman despite resolving jaundice. The frequency of jaundice was 1 in 78 209 co-amoxiclav prescriptions. Liver biopsy, available in 12 patients, showed perivenular bilirubinostasis, accompanying reactive ceroid laden macrophages, and portal inflammation with focal injury to interlobular bile ducts. Fourteen of 20 patients had DRB1*1501 compared with 27 of 134 controls (p<2.5 x 10(-6); odds ratio (OR) 9.25; relative risk (RR) 6.43). Of these, seven patients were homozygous for DRB1*1501(p< 10(-8); OR 35.54; RR=8.68) compared with two of 134 controls. All patients with DRB1*1501 had the extended haplotype DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602. There were no clinical or histological differences between genotypes. CONCLUSIONS: Co-amoxiclav associated hepatotoxicity may have a genetic basis and be delayed, severe, and prolonged, although complete recovery is usual.