Biomarker testing in oncology - Requirements for organizing external quality assessment programs to improve the performance of laboratory testing: revision of an expert opinion paper on behalf of IQNPath ABSL.

In personalized medicine, predictive biomarker testing is the basis for an appropriate choice of therapy for patients with cancer. An important tool for laboratories to ensure accurate results is participation in external quality assurance (EQA) programs. Several providers offer predictive EQA programs for different cancer types, test methods, and sample types. In 2013, a guideline was published on the requirements for organizing high-quality EQA programs in molecular pathology. Now, after six years, steps were taken to further harmonize these EQA programs as an initiative by IQNPath ABSL, an umbrella organization founded by various EQA providers. This revision is based on current knowledge, adds recommendations for programs developed for predictive biomarkers by in situ methodologies (immunohistochemistry and in situ hybridization), and emphasized transparency and an evidence-based approach. In addition, this updated version also has the aim to give an overview of current practices from various EQA providers.

Occupational exposure to pesticides and bile tract carcinoma in men: results from a European multicenter case-control study.

OBJECTIVES: To estimate the associations between occupational exposure to pesticides and extrahepatic biliary tract carcinoma in men, a population-based case-control study was carried out. METHODS: Cases (n = 104), aged 35-70, diagnosed in 1995-1997, were sampled by active reporting systems from hospitals. Controls (n = 1,401) were a random sample of the general male population. Information on occupation and confounding factors was obtained by questionnaires. Exposures were quantified with respect to time, application methods, and use of personal protective equipment. Intensity was evaluated by using a published algorithm which weighted the exposure assigned according to the use of personal protective equipment and mode of application. Logistic regression analyses were conducted adjusted for gallstones, age, and country. RESULTS: Being ever exposed to pesticides resulted in an odds ratio (OR) of 1.0 [95%-confidence interval (CI) 0.6-1.6]. A modestly elevated risk was found for backpack mounted sprayers OR = 1.4 [95% CI 0.7-2.6] and vine farmers OR = 2.5 [95% CI 0.9-7.2]. Using time periods and exposure frequency as intensity measure, no elevated risks were found. The only exception was year of maximum exposure which yielded an OR of 1.6 [95% CI 0.7-3.5]. However, no clear trend was observed in this analysis. CONCLUSIONS: This study does not rule out that pesticide exposure represents an occupational risk factor for extrahepatic biliary tract carcinoma, but no indication of a strong association was observed. Some modes of exposure were weakly, albeit not significantly associated with carcinoma risk. The observed estimates of effects may be influenced by a lack of precise exposure assessment. Different chemical compositions of pesticides were utilized during a long time span of pesticide exposure, and it should be considered that the exposure is assessed with substantial uncertainty that could non-differential and bias results toward the null.

Polymorphic MLH1 and risk of cancer after methylating chemotherapy for Hodgkin lymphoma.

BACKGROUND AND OBJECTIVE: Methylating agents are effective chemotherapy agents for Hodgkin lymphoma, but are associated with the development of second primary cancers. Cytotoxicity of methylating agents is mediated primarily by the DNA mismatch repair (MMR) system. Loss of MLH1, a major component of DNA MMR, results in tolerance to the cytotoxic effects of methylating agents and persistence of mutagenised cells at high risk of malignant transformation. We hypothesised that a common substitution in the basal promoter of MLH1 (position -93, rs1800734) modifies the risk of cancer after methylating chemotherapy. METHODS: 133 patients who developed cancer following chemotherapy and/or radiotherapy (n = 133), 420 patients diagnosed with de novo myeloid leukaemia, 242 patients diagnosed with primary Hodgkin lymphoma, and 1177 healthy controls were genotyped for the MLH1 -93 polymorphism by allelic discrimination polymerase chain reaction (PCR) and restriction fragment length polymorphism assay. Odds ratios and 95% confidence intervals for cancer risk by MLH1 -93 polymorphism status, and stratified by previous exposure to methylating chemotherapy, were calculated using unconditional logistic regression. RESULTS: Carrier frequency of the MLH1 -93 variant was higher in patients who developed therapy related acute myeloid leukaemia (t-AML) (75.0%, n = 12) or breast cancer (53.3%. n = 15) after methylating chemotherapy for Hodgkin lymphoma compared to patients without previous methylating exposure (t-AML, 30.4%, n = 69; breast cancer patients, 27.2%, n = 22). The MLH1 -93 variant allele was also over-represented in t-AML cases when compared to de novo AML cases (36.9%, n = 420) and healthy controls (36.3%, n = 952), and was associated with a significantly increased risk of developing t-AML (odds ratio 5.31, 95% confidence interval 1.40 to 20.15), but only in patients previously treated with a methylating agent. CONCLUSIONS: These data support the hypothesis that the common polymorphism at position -93 in the core promoter of MLH1 defines a risk allele for the development of cancer after methylating chemotherapy for Hodgkin lymphoma. However, replication of this finding in larger studies is suggested.

The established and future biomarkers of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an asbestos-related cancer with a median survival of 12months. The MPM incidence is 1-6/100,000 and is increasing as a result of historic asbestos exposure in industrialized countries and continued use of asbestos in developing countries. Lack of accurate biomarkers makes diagnosis, prognostication and treatment prediction of MPM challenging. The aim of this review is to identify the front line of MPM biomarkers with current or potential clinical impact. Literature search using the PubMed and PLoS One databases, the related-articles function of PubMed and the reference lists of associated publications until April 26th 2015 revealed a plethora of candidate biomarkers. The current gold standard of MPM diagnosis is a combination of two positive and two negative immunohistochemical markers in the epithelioid and biphasic type, but sarcomatous type do not have specific markers, making diagnosis more difficult. Mesothelin in serum and pleural fluid may serve as adjuvant diagnostic with high specificity but low sensitivity. Circulating proteomic and microRNA signatures, fibulin-3, tumor cell gene-ratio test, transcriptomic, lncRNA, glycopeptides, pleural fluid FISH assay, hyaluronate/N-ERC mesothelin and deformability cytometry may be important future markers. Putative predictive markers for pemetrexed-platinum are tumor TS and TYMS, for vinorelbine the ERCC1, beta-tubuline class III and BRCA1. Mutations of the BAP1 gene are potential markers of MPM susceptibility. In conclusion, the current status of MPM biomarkers is not satisfactory but encouraging as more sensitive and specific non-invasive markers are emerging. However, prospective validation is needed before clinical application.

The increasing incidence and prevalence of eosinophilic oesophagitis outpaces changes in endoscopic and biopsy practice: national population-based estimates from Denmark.

BACKGROUND: National population-based medical registries in Denmark offer a unique opportunity to study eosinophilic oesophagitis (EoE) epidemiology. AIM: To determine the incidence and prevalence of EoE in Denmark, and evaluate whether an increase in endoscopy with biopsy activity explains changes in these trends. METHODS: The Danish National Pathology Registry, Danish National Patient Registry and Danish Registry of Medicinal Product Statistics were queried from 1997 to 2012. Using an EoE case-finding algorithm validated for Danish patients, EoE cases were identified during each year of the study period; we also identified all patients with oesophageal eosinophilia. Using the known population of Demark, the annual incidence and prevalence of EoE were determined. We also determined the number of oesophageal biopsies performed each year in Denmark, and compared the change in the incidence rate to the change in biopsy rate. RESULTS: Between 1997 and 2012, 1708 patients had oesophageal eosinophilia, of whom 844 met the case definition of EoE. There were seven new cases of EoE in 1997 and 145 new cases in 2012, corresponding to a 19.5-fold increase in incidence (0.13/100 000 to 2.6/100 000). There were 769 total cases in 2012 (prevalence of 13.8/100 000). Over the same time frame, the oesophageal biopsy rate increased only 1.9 fold, from 91.1/100 000 to 175.3/100 000. CONCLUSIONS: The incidence and prevalence of EoE markedly increased in Denmark over the past 15 years. This increase far outpaced the increase in oesophageal biopsy utilisation, indicating that changes in the frequency of EoE are not due to changes in biopsy rates alone.

Mutations in the tumor suppressor gene p53 in human liver cancer induced by alpha-particles.

The p53 tumor suppressor gene is mutated in varying fractions of almost all tumor types studied. The rate of mutations and the mutational spectrum in some tumors are specific for environmental mutagens assumed to be involved in the carcinogenic process. Thus, hepatocellular carcinomas supposedly induced by aflatoxin exposure often contain a specific point mutation in codon 249, and in lung cancers of miners with heavy radon exposure, another specific point mutation in codon 249 suggestive of an alpha-particle-specific mutation has been shown. The interpretation of studies linking the mutational spectrum with specific environmental exposures is complicated by the multifactorial or unknown genesis of most tumors. However, people given injections of the X-ray contrast medium Thorotrast (Th) in the past have experienced an enormous risk of liver tumors, and virtually all of these are supposedly induced by alpha-particles from the decay of 232Th. The examination of these tumors may provide evidence as to whether specific p53 point mutations are relevant in alpha-particle carcinogenesis. Therefore, we collected paraffin-embedded, formalin-fixed archival tissues from 18 hepatocellular carcinomas, 9 cholangiocarcinomas, and 9 hepatic angiosarcomas from Thorotrast-exposed patients. The tissues were analyzed for p53 protein expression by immunohistochemical staining by using the mAb DO-7 and for mutations of exons 5-8 by PCR and constant denaturant gel electrophoresis. G --> T transversions of the third base of codon 249 of the p53 gene were specifically screened for by restriction enzymes. No high score for p53 protein expression (i.e., positive staining of >20% of examined cells) was observed; lower scores were seen in 5 of 18 (28%) hepatocellular carcinomas, 1 of 9 (11%) cholangiocarcinomas, and 0 of 8 (0%) hepatic angiosarcomas. Only one p53 mutation, a heterozygous T --> G transversion of the first base codon 176, occurred in a hepatocellular carcinoma. The rate of p53 point mutations in alpha-particle-induced liver tumors seems to be lower than in European hepatocellular carcinomas in general. The study does not exclude the possibility that alpha-particle carcinogenesis may involve inactivation of p53 by gross deletions of the gene, but it speaks against the proposed specificity of point mutations of codon 249 in cancer supposedly induced by alpha-particles from radon progeny.

Combined adrenal myelolipoma and adenoma associated with Cushing's syndrome.

A case of Cushing's syndrome in a 31-year-old woman is presented. The resected left adrenal gland revealed a tumor consisting of cortical cells intermingled with myelolipomatous tissue comparable to that of normal bone marrow. The adjacent cortex was atrophic. Postoperative plasma cortisol concentrations have remained quite low. Previously, 28 cases of surgically removed adrenocortical tumors with a main diagnosis of myelolipoma have been published. Of these, three cases (two pituitary Cushing's disease, one steroid 21-hydroxylase deficiency) were associated with endocrine dysfunction. The combination of a myelolipoma and a true adenoma has only been described once before (in a case of virilization) and never in connection with Cushing's syndrome. The etiology of myelolipoma is discussed, and a local trigger mechanism related to adrenocortical growth disturbances is suggested.

Primary liver tumors among Danish patients exposed to Thorotrast.

The potential carcinogenic effects of internally deposited alpha-particle-emitting nuclides, notably plutonium, in the liver in humans are unknown but are of concern in relation to exposures from the nuclear industry. However, patients injected with the radiographic contrast medium Thorotrast are chronically exposed to alpha-particle radiation from 232ThO2 in the liver. Among 1003 patients injected with Thorotrast, 584 of whom were alive 15 years after the injection and 40 at the end of follow-up, a total of 127 liver cancers were diagnosed, 45 of which were hepatocellular carcinomas, 41 cholangiocarcinomas and 33 hemangiosarcomas. The median time from injection to diagnosis was 35 years (range 18-48) and the cumulative frequency was 55.4% after 48 years. In univariate and multivariate analyses, the cumulative frequency of liver cancer was best described as a function of the estimated mean cumulative alpha-particle radiation dose to the liver 15 years ago, being independent of age, gender and volume of injected Thorotrast. This may be interpreted to mean that the liver cancer rate is not related to the dose rate and that the period from malignant transformation to diagnosis of cancer is 15 years. The risk of liver carcinogenesis induced by alpha-particle radiation, assuming 15 years from induction to diagnosis, was estimated to be 712 cases/10(4) persons per gray. This value is considerably higher than estimated earlier.

Histological sampling with a 23 gauge modified Menghini needle.

A 23 gauge modified Menghini (Surecut) needle biopsy technique for obtaining tissue core biopsies was compared with the conventional fine needle aspiration biopsy technique in the diagnosis of ultrasonically detected abdominal mass lesions. In 30 consecutive cases (19 malignant and 11 benign), adequate material for histological examination was obtained in 87% and for cytological examination in 97%. The diagnostic accuracy with respect to malignancy was 84% for histology and 89% for cytology. The predictive value of malignancy was 100% in both. The histological material provided additional information in four cases of malignancy, concerning the type and origin of the tumours, and in nine cases of benign lesions, indicating the type of lesion that appeared as a tumourlike mass in the ultrasound study. The Surecut needle biopsy has been found valuable in obtaining histological material from abdominal mass lesions and may supplement or even replace the fine needle aspiration biopsy in such lesions.

Primary leptomeningeal T-cell lymphoma: a case and a review of primary T-cell lymphoma of the central nervous system.

A case of primary leptomeningeal T-cell lymphoma of pleomorphic large cell type is presented. The lymphoma, which was first diagnosed at autopsy, occurred in a 67-year-old man, who had no personal or family history of immunodeficiency disorders. Review of the literature reveals only 13 immunocytochemically well-documented cases of primary T-cell lymphoma in the CNS. In five of these cases the lymphoma was apparently confined to the leptomeninges. The relatively high frequency of leptomeningeal presentation may indicate that this tumor form is more common with primary T-cell lymphoma of the CNS than with B-cell neoplasms.

[Autoimmune hepatitis. Forms of manifestation, diagnosis and treatment]. / Autoimmun hepatitis. Fremtroedelsesformer, diagnostik og behandling.

A retrospective study concerning ten patients with autoimmune hepatitis (AiH), diagnosed during a 2 1/2-year period is presented. The age of the patients ranged from 25 to 82 years and nine of the patients were women. Their symptoms included jaundice, pruritus, fever, anorexia and fatigue during a few weeks to years. Seven patients had increased serum aspartate aminotransferase (ASAT) levels. The three patients with normal ASAT levels had hypoalbuminaemia, decreased level of prothrombin or high levels of serum immunoglobulin G. Moderate or high levels of smooth muscle antibody titer were detected in nine patients, while none had increased levels of anti-nuclear antibody titer. Histological features of moderate or severe chronic active hepatitis were demonstrated in nine patients. One patient presented with clinical and histological features of acute hepatitis. Prednisolone therapy was followed by biochemical improvement in all the patients. In one patient, maintenance therapy with prednisolone was combined with azathioprine.

[Hepatocellular adenoma after oral contraception]. / Hepatocellulaert adenom efter peroral kontraception.

Raised serum basic phosphatase was found incidentally in a woman aged 43 years. Investigation with biopsy revealed a hepatocellular adenoma. The tumour regressed after withdrawal of Neogentrol oral contraception which the patient had consumed for 17 years. The patient did not desire invasive treatment. Employment of oral contraceptive steroids for more than two years is associated with increased occurrence of hepatocellular adenomata. The hepatocellular adenoma is a clearly delimited, most frequently solitary, benign tumour with limited malignant potential but with a considerable risk of rupture with haemorrhage even after withdrawal of oral contraception. The hepatocellular adenoma has no malignant tumour vessels (in contrast to hepatocellular carcinoma) and it appears as a cold region on scintigraphy (in contrast to focal nodular hyperplasia), but the diagnosis can only be established with certainty by histological examination. The hepatocellular adenoma consists most frequently of large pale hepatocytes in trabeculae surrounded by a net of reticulin and separated by sinusoids. Biliary passage and portal spaces do not occur. The best treatment consists of excision of the tumour or embolisation. If invasive treatment is postponed, regular scanning should be performed to observe regression or progression of the tumour and oral contraception and pregnancy should be advised against on account of the risk of growth of the tumour.

Neuronal features of oligodendrogliomas--an ultrastructural and immunohistochemical study.

AIMS: To assess neuronal differentiation in oligodendrogliomas (ODGs). METHODS AND RESULTS: An electron microscopic and immunohistochemical study of 41 consecutive cases was performed. In all cases, tumour cells with neuritic structures were identified ultrastructurally, including synapses and neurosecretory granules. For the immunohistochemical identification of synaptophysin, monoclonal antibody clones 27G12, Snp88 and SY38 and a polyclonal antibody were compared in optimized protocols on slides from a spectrum of tissues and 16 ODGs. 27G12 gave the best signal-to-noise ratio, while SY38 gave the poorest. When 27G12 was applied on all 41 ODGs, widespread immunoreactivity was obtained in 100%. Among three antibodies to chromogranin compared similarly, clone LK2H10 and a polyclonal antibody gave identical patterns of immunoreactivity, whereas clone DAK-A3 gave weaker reactions. When LK2H10 was applied on all tumours, staining was found in 12 (29%). All tumours but one stained strongly for glial fibrillary acidic protein and all for synapsin I. Fluorescence in situ hybridization analysis showed a concomitant 1p/19q deletion in 12/16 ODGs. CONCLUSIONS: Our study provides evidence for widespread neuronal differentiation in ODGs, suggesting that these tumours may be derived from progenitor cells with limited commitment. Antibody selection and protocol optimization are mandatory for reliable immunohistochemistry results.

Diverticular bile duct lesion in chronic active hepatitis.

Liver needle biopsies from patients with non-A, non-B chronic active hepatitis and so-called abnormal bile duct epithelium were studied with a three-dimensional method. Photographs of bile duct structures in serial sections were transferred to acrylic plates. Five bile duct lesions of a not previously described diverticular type were revealed. The diverticuli were of varying shape with a diameter of 30 to 110 microns and a length of 75 to 150 microns budding from small (12 to 25 microns), slightly ectatic bile ducts. The diverticular epithelium was disordered. Some cells appeared as bile duct cells, but most were larger, with rounded nuclei, prominent nucleoli and abundant eosinophilic cytoplasm, sometimes with periodic acid-Schiff-positive, diastase-resistant granules. The lesions were only partly surrounded by a basement membrane. They were all embedded in a tight mononuclear inflammatory infiltrate associated with pronounced periportal piecemeal necrosis. In two cases, a germinal center was adjacent to the epithelium. The pathogenesis of the diverticular bile duct lesion is unknown, but the diverticuli probably represent Hering ducts and groups of periportal liver cells which have escaped the piecemeal necrosis.

Abnormal bile duct epithelium accompanying septicaemia.

A 56-year-old female without previous hepatobiliary disease developed a severe obstructive cholestasis following E. coli urinary tract infection with septicaemia. Liver biopsy showed cholangitis and a unique abnormality of almost all the interlobular bile ducts; the epithelium was irregular with polymorphic, angular, and hyperchromatic or pyknotic nuclei. Some ducts were ectatic , others narrowed due to protrusion of proliferating epithelium. In some areas the ducts were blurred or completely destroyed. Cholangitis or granulomas were, however, not present. Abnormal interlobular bile ducts have to our knowledge not previously been described in septicaemia. The lesion is morphologically distinguishable from other types of abnormal bile ducts. It is considered to be caused by endotoxaemia and seems to be reversible. The cholestasis may be due to endotoxic alteration of biliary secretion, bacterially induced inspissation of bile, and/or mechanical obstruction to the bile duct lesions.

Ubiquitin: an immunohistochemical marker of Mallory bodies and alcoholic liver disease.

One hundred forty eight liver needle biopsies, comprising 88 consecutive biopsies from patients with clinically diagnosed or suspected alcoholic liver disease and 60 selected biopsies from non-alcoholics, were immunostained for the cell stress protein ubiquitin (Ub). Ub + cells were detected in all of 33 biopsies with alcoholic hepatitis (AH). Practically all Mallory bodies (MBs) showed intense Ub-staining. In addition, many cells revealed Ub + granules lying aggregated (pre-MBs) or dispersed in the cytoplasm of ballooned cells. The mean number of Ub + cells in 10 biopsies with AH was more than 30 times the number of MBs and more than 6 times the number of MBs + pre-MBs found in adjacent Haematoxylin-Eosin (HE) stained sections. Among 55 biopsies from alcoholic patients without AH (i.e. without MBs or pre-MBs in HE stained sections), Ub + cells occurred in eight (14.5%). Among the 60 selected biopsies from non-alcoholic patients with liver diseases in which hepatocyte ballooning and occasionally MBs are seen, a few Ub + cells were revealed in two out of ten with primary biliary cirrhosis (one of which also showed MBs) but none in biopsies with primary sclerosing cholangitis, long-standing large duct obstruction, or various hepatitides. Thus Ub-immunostaining appears to be a highly sensitive and specific method in the detection of MBs and MB precursor stages, making it a valuable tool in the study of alcoholic liver disease, and particularly a more objective method (compared to conventional staining methods) of diagnosing alcoholic hepatitis.

Focal fatty change of the liver. A review and a case associated with continuous ambulatory peritoneal dialysis.

Focal fatty change (FFC) of a liver with unequivocal normal architecture is rare. A review of the literature revealed 39 histologically well-documented cases. Well-known steatogenic conditions were present in most of these cases. Focal ischaemia or a varying rate of mobilization of fat in the liver have been suggested as a cause of the focality of the lesions. FFC occurring in a diabetic patient on continuous ambulatory peritoneal dialysis (CAPD) is presented. The FFC in this and in ten previously reported cases associated with CAPD and intraperitoneal insulin therapy had a unique subcapsular distribution, which may suggest a specific pathogenetic mechanism involving insulin. The clinical significance of FFC in the differential diagnosis from other fatty lesions of the liver is summarized.

Pattern of progression in liver injury following jejunoileal bypass for morbid obesity.

Liver biopsies from 34 patients with morbid obesity, performed before and 5-9 months after jejunoileal bypass, were studied. The patients were divided into four groups according to preoperative findings: A: no or slight steatosis (15 patients), B: moderate-severe steatosis (6), C: steatohepatitis (steatosis + lobular lymphocytic inflammation) (8), D: steatofibrosis (steatosis + pericellular fibrosis) (5). In Group A, 12 patients showed postoperative progression to either moderate/severe steatosis, steatohepatitis, or steatofibrosis. In Group B, all patients progressed to steatohepatitis or steatofibrosis, and one developed septate fibrosis. All patients in Group C progressed to steatofibrosis, and 5 developed septate fibrosis or cirrhosis. In Group D, 3 developed bridging fibrosis. Mallory bodies appeared postoperatively in 11 patients (32%), all of whom preoperatively had either severe steatosis, steatohepatitis, or steatofibrosis. Only patients with postoperative pericellular fibrosis and Mallory bodies developed deranged architecture: 6 septate/bridging fibrosis, and 3 cirrhosis. Five patients, all with deranged architecture, developed reversible liver insufficiency. Progressive liver injury after jejunoileal bypass appears to reflect aggravation of a pre-existing liver lesion. The sequence of events: increasing steatosis, lobular lymphocytic inflammation, pericellular fibrosis, Mallory bodies, and deranged architecture is similar to that of the alcoholic liver lesion, indicating common pathogenetic mechanisms.