JCP Online First

Comparison of the Pathvysion and Poseidon HER2 Fish Assays in Measuring HER2 Amplification in Breast Cancer-a Validation Study

Thu, 03 Dec 2009 21:17:18 PST

Aims: The current study was done as a validation study prior to setting up a clinical HER2 testing service using the new commercial Poseidon HER2 FISH assay. However, it was felt that the experience of the authors of this study may be of interest to other laboratories when considering setting up a HER2 diagnostic facility.

Methods: One hundred and twenty-two patients who had been diagnosed with invasive breast cancer were selected. Immunolabeling with Herceptest, Pathvysion, and Poseidon FISH assays were carried out using tissue microarray blocks.

Results: Concordance correlation coefficients showed near perfect agreement in average HER2 and centromere specific signal counts per cell and in the HER2/CEP17 ratios between the PathVysion and the Poseidon FISH assays. In addition, the Kappa measure showed perfect agreement (Kappa=0.9441, p<0.0001), and - if only 2+ cases were considered - substantial agreement (Kappa=0.7671, p=0.0006) between the two assays. The sensitivity and the specificity of the Poseidon FISH kit were calculated to be 95.2% and 100%, respectively, whereas the positive (PPV) and negative predictive values (NPV) were 100% and 99%. With regard to the ability to presume HER2 polysomy, the Poseidon FISH kit had a sensitivity of 93.3% and a specificity of 99.1% with a PPV and NPV of 93.3% and 99.1%, respectively, as assessed with PathVysion classification as reference.

Conclusions: Statistical analysis confirmed that the two FISH assays are comparable in terms of detection of HER2 gene amplification. Proceeding from these findings, the genetic diagnoses obtained with the Poseidon kit can be considered as valuable as the results from the Food and Drug Administration (FDA) approved PathVysion assay, and propose its utilization in routine HER2 diagnostics.

Microbial infections in eight genomic subtypes of Chronic Fatigue Syndrome / Myalgic Encephalomyelitis (CFS/ME)

Wed, 02 Dec 2009 16:43:02 PST

We have previously reported genomic subtypes of CFS/ME based on expression of 88 human genes. In this study we attempted to reproduce these findings, determine specificity of this signature to CFS/ME, and test for associations between CFS/ME subtype and infection.

We determined expression levels of 88 human genes in blood of 61 new patients with idiopathic CFS/ME (according to Fukuda criteria), 6 patients with Q-fever associated CFS/ME form the Birmingham Q-fever outbreak (according to Fukuda criteria), 14 patients with endogenous depression (according to DSM-IV criteria) and 18 normal blood donors. In patients with CFS/ME differential expression was confirmed for all 88 genes. Q-CFS/ME patients had similar patterns of gene expression to idiopathic CFS/ME. Gene expression in endogenous depression patients was similar to that in the normal controls, except for upregulation of five genes (APP, CREBBP, GNAS, PDCD2, PDCD6).

Clustering of combined gene data in CFS/ME patients for this and our previous study (n=117 CFS/ME patients) revealed genomic subtypes with distinct differences in SF-36 scores, clinical phenotypes, severity and geographical distribution. Antibody testing for Epstein-Barr virus (EBV), enterovirus, Coxiella burnetii and parvovirus B19 revealed subtype-specific relationships for EBV and enterovirus, the two most common infectious triggers of CFS/ME.

Pharmacological activation of the p53 pathway in haematological malignancies

Saha, M. N, Chang, H. — Wed, 02 Dec 2009 16:43:01 PST

p53 gene mutations are rarely detected at diagnosis in common hematologic cancers such as multiple myeloma (MM), acute myeloid leukaemia (AML), chronic lymphocytic leukaemia (CLL), and Hodgkin’s disease (HD), although their prevalence may increase with progression to more aggressive or advanced stages. Therapeutic induction of p53 might therefore be particularly suitable for the treatment of hematologic malignancies. Some of the anti-tumour activity of current chemotherapeutics has been derived from activation of p53. However, until recently it was unknown if p53 signaling is functional in some of the hematologic cancers including multiple myeloma (MM) and if p53 activity is sufficient to trigger an apoptotic response. With the recent discovery of nutlins, which represent the first highly selective small molecule inhibitors of the p53-MDM2 interaction, pharmacologic tools are now available to induce p53 irrespective of upstream signaling defects, and to functionally analyze the downstream p53 pathway in primary leukaemia and lymphoma cells. Combination therapy is emerging as a key factor, and development of non-genotoxic combinations seems very promising for tackling the problems of toxicity and resistance. This review will highlight recent findings in the research into molecules capable of modulating p53 protein activities and mechanisms that activate the p53 pathway restoring response to therapy in hematological malignancies.

Postmortem Candidemia: Marker of Disseminated Disease

Wed, 25 Nov 2009 01:43:45 PST

Aim: The significance of finding Candida species in heart blood cultures obtained at postmortem examination has never been studied. Therefore, we describe the findings of autopsy patients with postmortem candidemia and compare them to autopsy patients with antemortem candidemia.

Method: Twenty-three patients with Candida species isolated from heart blood at autopsy were identified over a ten-year period. These patients were compared to 10 autopsy patients found during the same time period with antemortem blood cultures isolating Candida species, but not positive postmortem heart blood cultures. Ante- and postmortem records were reviewed.

Results: All the 23 patients with Candida species isolated from postmortem blood culture all had one or more antemortem risk factors for disseminated candidiasis such as positive antemortem blood cultures, isolation of Candida from sterile internal sites, neutropenia, recent abdominal surgery, broadspectrum antibiotic administration or the use of central venous catheters or other invasive devices. Eight patients had histologic proof of invasive candidiasis in addition to the positive heart blood cultures. This group did not differ with respect to risk factors from 10 autopsy patients with disseminated candidiasis and antemortem blood cultures with Candida species. However, all the patients with antemortem candidemia had histologic evidence of disseminated candidiasis at autopsy.

Conclusion: Candidemia, when documented by heart blood culture performed at autopsy or by antemortem blood culture, is an insensitive, but highly specific indicator of disseminated candidiasis.

A case of Chronic Fatigue Syndrome following H1N1 influenza (swine influenza)

Vallings, R. — Mon, 26 Oct 2009 17:11:21 PDT

Case report of an adolescent boy who was diagnosed as suffering from Chronic Fatigue Syndrome 5 months after infection with H1N1 influenza.

Recent advances in understanding the molecular basis of Paget's disease of bone

Goode, A., Layfield, R. — Mon, 26 Oct 2009 17:09:23 PDT

Paget’s disease of bone (PDB) is a relatively common disorder characterised by increased bone turnover within discrete lesions throughout the skeleton. The condition has a strong genetic component, with mutations affecting the SQSTM1 gene which encodes the p62 protein often found in PDB patients, although environmental factors also play an important role in disease aetiology. The precise disease mechanism(s) both in familial and sporadic forms of PDB is unclear, although defective RANK-NF-kB signalling has been suggested to contribute to the increased activity of pagetic osteoclasts in the former. Here, we review recent advances in the understanding of the molecular basis of PDB with particular emphasis on findings since 2008, and focus on newly defined functions of the p62 protein upon which SQSTM1 mutations may impact in the development of the pagetic phenotype.

Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens

Lee, S. H., Vigliotti, V. S, Pappu, S. — Mon, 26 Oct 2009 17:06:26 PDT

Aims:

Persistent infection indicated by detection of human papillomavirus-16 (HPV-16) on repeat testing over a period of time poses the greatest cervical cancer risk. However, variants of HPV-16, HPV-31 and HPV-33 may share several short sequence homologies in the hypervariable L1 gene commonly targeted for HPV genotyping. The purpose of this study was to introduce a robust laboratory procedure to validate HPV-16 detected in clinical specimens, using the GenBank sequence database as the standard reference for genotyping.

Methods:

A nested polymerase chain reaction (PCR) with two pairs of consensus primers was used to amplify the HPV DNA released in crude proteinase K digestate of the cervicovaginal cells in liquid-based Papanicolaou cytology specimens. The positive nested PCR products were used for direct automated DNA sequencing.

Results:

A 48-base sequence downstream of the GP5+ priming site or a 34-base sequence upstream thereof was needed for unequivocal validation of an HPV-16 isolate. Selection of a 45-base, or shorter, sequence immediately downstream of the GP5+ site for Basic Local Alignment Search Tool (BLAST) sequence analysis invariably led to ambiguous genotyping results.

Conclusions:

DNA sequence analysis may be used for differential genotyping of HPV-16, HPV-31 and HPV-33 in clinical specimens. However, selection of the signature sequence for BLAST algorithms is crucial to distinguish certain HPV-16 variants from other closely related HPV genotypes.

Platelet contamination causing Staphylococcus aureus septicaemia

Coutinho, H., Galloway, A., Adjukiewicz, K., Cleeve, V. — Mon, 26 Oct 2009 17:05:06 PDT

Platelet transfusions have the highest incidence of post transfusion sepsis compared to any other blood products.1 Recent reports suggest that platelet related bacteraemia occurs at a frequency approximately 50 times greater than that for red blood cells.2 The source is usually skin contaminants from the donor and several organisms have been implicated, the commonest of which are coagulase negative staphylococci. We present a case of serious Staphylococcus aureus sepsis following platelet transfusion and discuss relevant methods to detect and prevent bacterial contamination.

Acute enterovirus infection followed by myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and viral persistence.

Chia, j. K, Chia, A. Y, Voeller, M., Lee, T. M, Chang, R. — Wed, 14 Oct 2009 17:23:10 PDT

A number of infections have been associated with myalgia encephalomyelitis/chronic fatigue syndrome (ME/CFS). Often occurring in epidemics, enteroviruses are significant causes of respiratory, gastrointestinal infections, non-specific flu-like illnesses, and many disseminated infections. It has been difficult to demonstrate causality for chronic diseases without having well-documented cases of acute enterovirus infections. In this report, we described 3 patients presenting with acute enterovirus infections, which were followed by ME/CFS, and the persistent viral infections were demonstrated by finding enterovirus VP1 protein and RNA in the stomach.

Enhanced BMS cut up role in colonic cancer reporting

Wed, 16 Sep 2009 02:07:26 PDT

Aims: To extend the BMS cut up role to include gastrointestinal category D colorectal cancer resection specimens and to address issues of quality and safety by presenting performance data from the first 50 BMS cut up specimens in comparison to national guidelines and pathologist performance over the same timeframe.

Methods: Close mentoring and consultant supervision was carried out for every case with adherence to standard operating procedures and following colorectal cancer dataset guidelines as published by the RCPath. Performance targets were audited including anticipated spread of Duke’s stage, targets for mean lymph node harvest, percentage extramural vascular invasion and serosal involvement, and mean tumour blocks sampled. Histological pre-reporting of 20 cases was encouraged and time spent by BMS and consultant at all stages of specimen reporting was noted.

Results: Performance targets were all exceeded by the BMS and compared favourably with pathologist performance. A measure of consultant cut up and histology reporting time saved was identified.

Conclusions: Benefits of extending the BMS role to category D specimens may include BMS professional advancement, efficient use of consultant time, and development of a team approach to cancer reporting. Achievement of colorectal cancer performance targets and favourable comparison with pathologist performance implies there was no perceived detrimental effect on quality or safety and hence patient management.

The pathology of Wilms' tumour (nephroblastoma): The International Society of Paediatric Oncology approach

Vujanic, G. M, Sandstedt, B. — Sun, 16 Aug 2009 22:45:16 PDT

In the International Society of Paediatric Oncology renal tumour trials, pre-operative chemotherapy has been successfully applied with resulting reduction of tumour rupture and increased favourable stage distribution of nephroblastoma. Post-operative treatment includes chemotherapy and sometimes radiotherapy in a risk-adapted approach based on histological sub-classification and stage of the tumour. However, preoperative chemotherapy alters tumour’s histological features and distribution of subtypes, and makes staging more difficult. The paper highlights the most common practical diagnostic difficulties that a pathologist is faced with in dealing with pre-treated nephroblastomas. It emphasises the importance of a systematic, step-by-step analysis based on adequately sampled material, in order to accurately sub-classify a nephroblastoma as a low, intermediate or high risk tumour and assign its genuine stage. Finally, it outlines the standard operating procedure for submission of renal tumours for rapid central pathology review which allows the treating oncologists to apply the optimal treatment protocol.

Tracking the footprints of the rabies virus: are we any closer to decoding this elusive virus?

Mahadevan, A., MS, S., SN, M., SK, S. — Thu, 09 Oct 2008 08:59:25 PDT

The authors have withdrawn the paper following an allegation of plagiarism

Molecular pathology of NUT midline carcinomas

French, C. A. — Fri, 13 Jun 2008 08:45:17 PDT

NUT midline carcinoma (NMC) is a rare, highly lethal cancer that occurs in children and adults of all ages. NMCs uniformly present in the midline, most commonly in the head, neck, or mediastinum, as poorly differentiated carcinomas with variable degrees of squamous differentiation. This tumor is defined by rearrangement of the Nuclear protein in testis (NUT) gene on chromosome 15q14. In most cases, NUT is involved in a balanced translocation with the BRD4 gene on chromosome 19p13.1, an event that creates a BRD4-NUT fusion gene. Variant rearrangements, some involving the BRD3 gene, occur in the remaining cases. NMC is diagnosed by detection of NUT rearrangement by fluorescence in situ hybridization or RT-PCR. Due its rarity and lack of characteristic histologic features, most cases of NMC currently go unrecognized.

Best Practice in Primary Care Pathology: review 3

Smellie, W. S. A — Fri, 19 May 2006 09:47:25 PDT

This third best practice review examines four series of common primary care questions in laboratory medicine: (i) 'minor' blood platelet count and haemoglobin abnormalities, (ii) diagnosis and monitoring of iron deficiency anaemia , (iii) secondary hyperlipidaemia and hypertriglyceridaemia and (iv) HbA1c and microalbumin use in diabetes. The review is presented in question-answer format, referenced for each question series. The recommendations represent a précis of guidance found using a standardised literature search of national and international guidance notes, consensus statements, health policy documents and evidence-based medicine reviews, supplemented by MEDLINE EMBASE searches to identify relevant primary research documents. They are not standards but form a guide to be set in the clinical context. Most are consensus rather than evidence-based. They will be updated periodically to take account of new information.