Next generation diagnostic molecular pathology: Critical appraisal of quality assurance in Europe

Tumor evaluation in pathology is more and more based on a combination of traditional histopathology and molecular analysis. Due to the rapid development of new cancer treatments that specifically target aberrant proteins present in tumor cells, treatment decisions are increasingly based on the molecular features of the tumor. Not only the number of patients eligible for targeted precision medicine, but also the number of molecular targets per patient and tumor type is rising. Diagnostic molecular pathology, the discipline that determines the molecular aberrations present in tumors for diagnostic, prognostic or predictive purposes, is faced with true challenges. The laboratories have to meet the need of comprehensive molecular testing using only limited amount of tumor tissue, mostly fixed in formalin and embedded in paraffin (FFPE), in short turnaround time. Choices must be made for analytical methods that provide accurate, reliable and cost‐effective results. Validation of the test procedures and results is essential. In addition, participation and good performance in internal (IQA) and external quality assurance (EQA) schemes is mandatory. In this review, we critically evaluate the validation procedure for comprehensive molecular tests as well as the organization of quality assurance and assessment of competence of diagnostic molecular pathology laboratories within Europe.     

Ischaemic colitis mimicking inflammatory bowel disease in a young adult receiving oral anticoagulation

Ischaemic colitis (IC) is the most frequent form of gastrointestinal ischaemia. Discrepancy between non-specific symptoms and objective findings is a hallmark of IC. Thus delay of diagnosis is common due to its often subtle and unpredictable presentation. In particular, the clinical symptoms and signs of IC can overlap with those of inflammatory bowel disease. We present a case of a young man with known factor-V-Leiden mutation in whom IC developed during effective therapy with oral anticoagulants, presenting with symptoms and endoscopic findings suggestive of inflammatory bowel disease.     

CDKN2A (INK4A-ARF) mutation analysis to distinguish cutaneous melanoma metastasis from a second primary melanoma

The histologic differential diagnosis between a second primary cutaneous melanoma and cutaneous melanoma metastasis in a patient with a previous history of melanoma can be very difficult. This case report describes the first application of CDKN2A mutation analysis for discriminating a cutaneous melanoma metastasis from a new primary melanoma. In 2005, we received a skin excision of the right arm of a 38-year-old female patient for second opinion. Histologically, we considered the lesion to be a melanoma. The patient had a history of superficial spreading melanoma in the right subclavicular region, with a Breslow thickness of 1.1 mm, in 1998. The morphology showed resemblance to the present melanoma on the right arm, but the differential diagnosis between metastasis or second primary melanoma could not be made with certainty based on histology alone. We decided to perform TP53 and CDKN2A mutation analysis on both tumors. Molecular analysis revealed that both the melanoma of 1998 and of 2005 contained an identical CDKN2A mutation (a deletion in exon 1alpha, c.95_112del (p.Leu32_Leu37del)), which was absent in normal control tissue of the patient, thereby excluding a germline mutation. TP53 mutations were absent in both tumors and in normal skin. Based on these molecular findings the present melanoma on the right arm was diagnosed as a metastasis. Seven months later the patient died of widespread metastatic disease confirming the metastatic nature of the lesion. This case illustrates that molecular analysis can contribute to the sometimes-difficult differentiation between a second primary melanoma and a melanoma metastasis.     

Variation in T-SPOT.TB Spot Interpretation between Independent Observers from Different Laboratories

T-SPOT.TB is a specific assay for the diagnosis of tuberculosis. The assay needs to be performed with freshly isolated cells, and interpretation requires training. T-SPOT.TB has been used in various clinical-epidemiological settings, but so far no studies have evaluated the effect of interobserver variation in test reading. Our aim was to evaluate variation between different observers in reading T-SPOT.TB results. The study was nested within an ongoing cohort study, in which part of the T-SPOT.TB had been performed with frozen material. Culture plates were read visually by four different observers from two laboratories and by two automated readers. Of 313 T-SPOT.TB assays, 235 were performed with fresh cells and 78 were performed with frozen cells. No significant difference was found between results obtained with fresh cells and those obtained with frozen cells. The percentage of positive results varied between readers by maximally 15%; five/six raters were within a 6% difference in positive results. Analysis of the observed interrater differences showed that some individuals systematically counted more spots than others did. Because test interpretation includes subtraction of background values, this systematic variance had little influence on interindividual differences. The test result as positive or negative varied between independent raters, mainly due to samples with values around the cutoff. This warrants further study regarding determinants affecting the reading of T-SPOT.TB.Roughly a century after the introduction of the tuberculin skin test (TST), the recent development of gamma interferon release assays (IGRA) for specific detection of infection with Mycobacterium tuberculosis has realized a new class of immunodiagnostic tests that have extensively been evaluated for detection both of active tuberculosis (TB) and of latent TB infection (1, 2, 4, 7). T-SPOT.TB and QuantiFERON-TB Gold in-tube are the commercially available and approved IGRA formats, being based on culture of isolated peripheral blood mononuclear cells (PBMCs) and of whole blood, respectively. Numerous studies that evaluated the use of IGRA have been published in the past several years, showing their particular value for detection of latent TB infection in populations with high rates of false-positive TSTs due to Mycobacterium bovis BCG vaccination or exposure to nontuberculous mycobacteria (3, 5). T-SPOT.TB is based on the enzyme-linked immunospot assay technique in which cells responding with gamma interferon production after antigen stimulation are visualized as spots, which must be enumerated. This can be done by use of an automated spot reader or by using a magnifying glass. The assay is performed in four wells with different stimulations: medium as a negative control, phytohemagglutinin as a positive control, and peptides of the TB-specific antigens ESAT-6 (panel A of the assay) and CFP-10 (panel B of the assay). One of the disadvantages of T-SPOT.TB is that it must be performed with fresh material, which may not always be convenient. As the assay is based on single-well culture for each stimulus, random variability cannot be detected. Another disadvantage is that counting the spots might lead to variation when results are read by different observers or automated readers.Thus far, no studies have addressed the interobserver variability of the T-SPOT.TB. In the present study, these issues were addressed by using material obtained within an ongoing cohort study in The Netherlands in which part of the T-SPOT.TB assay was performed with frozen material for logistical reasons (blood arriving in the laboratory on a Friday was frozen since the assay needed to be completed 20 h later). We evaluated the reading of the T-SPOT.TB plates in two laboratories by different observers and by two automated readers. Next, we compared results of T-SPOT.TB obtained with freshly isolated cells to those obtained with frozen and thawed cells.     

Microscale mechanical properties of single elastic fibers: The role of fibrillin–microfibrils

Micromechanical properties of single elastic fibers and fibrillin–microfibrils, isolated from equine ligamentum nuchae using chemical and enzymatic methods, were determined with atomic force microscopy (AFM). Young's moduli of single elastic fibers immersed in water, devoid of or containing fibrillin–microfibrils, were determined using bending tests. Bending freely suspended elastic fibers on a micro-channeled substrate by a tip-less AFM cantilever generated a force versus displacement curve from which Young's moduli were calculated. For single elastic fibers, Young's moduli in the range of 0.3–1.5 MPa were determined, values not significantly affected by the presence of fibrillin–microfibrils. To further understand the role of fibrillin–microfibrils in vertebrate elastic fibers, layers of fibrillin–microfibrils were subjected to nano-indentation tests. From the slope of the force versus indentation curves, Young's moduli ranging between 0.56 and 0.74 MPa were calculated. The results suggest that fibrillin–microfibrils are not essential for the mechanical properties of single vertebrate elastic fibers.     

The performance of human dental pulp stem cells on different three-dimensional scaffold materials

The aim of this study was to investigate the in vitro and in vivo behavior of human dental pulp stem cells (DPSCs) isolated from impacted third molars, when seeded onto different 3-dimensional (3-D) scaffold materials: i.e. a spongeous collagen, a porous ceramic, and a fibrous titanium mesh. Scaffolds were loaded with DPSC, and subsequently divided into two groups. The first group was cultured in osteogenic differentiation medium in vitro for 4 weeks. The second group of samples was implanted subcutaneously in nude mice for 6 or 12 weeks. Samples cultured in vitro were analyzed by scanning electron microscopy and RT-PCR for dentin sialophosphoprotein (DSPP) expression. In vivo samples were evaluated by histology, RT-PCR and immunohistochemistry. The results indicated that in vitro, cells developed abundant deposition of mineralized extracellular matrix (ECM) with expression of DSPP in all 3-D materials. The simultaneous implantation experiment showed formation of tissue that was DSPP positive in all three scaffolds materials. However, the aspect of the formed tissues in all scaffolds resembled more connective tissue than a dentin-like tissue. Limited calcification of the ECM was only seen in the ceramic scaffold. In both experiments, no other differences could be attributed to the different materials used. In conclusion, the in vivo behavior of DPSC and their relations with 3-D scaffold materials should be further studied before clinical use can be considered.     

Design-related bias in hospital fall risk screening tool predictive accuracy evaluations: systematic review and meta-analysis

INTRODUCTION: Fall risk screening tools are frequently used as a part of falls prevention programs in hospitals. Design-related bias in evaluations of tool predictive accuracy could lead to overoptimistic results, which would then contribute to program failure in practice. METHODS: A systematic review was undertaken. Two blind reviewers assessed the methodology of relevant publications into a four-point classification system adapted from multiple sources. The association between study design classification and reported results was examined using linear regression with clustering based on screening tool and robust variance estimates with point estimates of Youden Index (= sensitivity + specificity - 1) as the dependent variable. Meta-analysis was then performed pooling data from prospective studies. RESULTS: Thirty-five publications met inclusion criteria, containing 51 evaluations of fall risk screening tools. Twenty evaluations were classified as retrospective validation evaluations, 11 as prospective (temporal) validation evaluations, and 20 as prospective (external) validation evaluations. Retrospective evaluations had significantly higher Youden Indices (point estimate [95% confidence interval]: 0.22 [0.11, 0.33]). Pooled Youden Indices from prospective evaluations demonstrated the STRATIFY, Morse Falls Scale, and nursing staff clinical judgment to have comparable accuracy. DISCUSSION: Practitioners should exercise caution in comparing validity of fall risk assessment tools where the evaluation has been limited to retrospective classifications of methodology. Heterogeneity between studies indicates that the Morse Falls Scale and STRATIFY may still be useful in particular settings, but that widespread adoption of either is unlikely to generate benefits significantly greater than that of nursing staff clinical judgment.     

An educational programme for peer review groups to improve treatment of chronic heart failure and diabetes mellitus type 2 in general practice

RATIONALE, AIMS AND OBJECTIVES: Peer review groups are considered helpful for quality improvement in primary care. An interactive educational programme for small peer groups was developed, focusing on the implementation of newly developed treatment guidelines. The aim is to evaluate the effect of the programme on adherence to treatment guidelines in general practice. METHODS: A cluster randomized trial using a balanced incomplete block design was used; one arm received a programme on treatment of chronic heart failure (CHF), the other on hypertension treatment in diabetes mellitus type 2 (T2DM). A random sample of 10 CHF and 10 T2DM patients per GP was drawn, for whom data were extracted from electronic patient records 1 years before and 6 months after the intervention. The outcomes were prescribing of ACE inhibitors, and antihypertensive treatment in T2DM. The effect was analysed separately for both programmes using multilevel regression models. RESULTS: All 27 peer review groups in one region in the Netherlands were randomized, of which 16 participated. No significant effects were observed in the CHF group or in the T2DM group. The opportunity for change was limited, as only 53% of the CHF patients and 60% of the T2DM patients had a contact with their GP between the intervention and follow-up measurement. CONCLUSION: The peer review programme was not successful for changing the treatment of chronic patients, although the programme focused on dealing with barriers perceived by the participants. Not all problems perceived can be solved in a peer group discussion.     

Aggregate distribution in frontal and motor cortex in Huntington's disease brain

Insoluble protein aggregates have been considered a pathological hallmark of Huntington's disease and other polyglutamine disorders. In this study the number of aggregates was assessed in the superior frontal gyrus and motor cortex of seven Huntington's disease patients and was compared with the symptoms (motor/mood) these patients displayed during the course of the disease. Regardless of the pattern of symptoms present in the patients, there was a consistently higher number of nuclear and non-nuclear aggregates in the superior frontal gyrus than in the motor cortex. This suggests that there is a consistent regional difference in the density of aggregates and that this consistency is not reflected in the variable symptomatology between cases.