BMC Clinical Pathology - Latest articles

Pathology of Rituximab-Induced Kaposi Sarcoma Flare

Liron Pantanowitz, Klaus Fruh, Sharon Marconi, Ashlee V Moses and Bruce J Dezube — 2008-07-23

Background: Kaposi sarcoma (KS) flare may occur following therapy with corticosteroids, as part of the immune reconstitution inflammatory syndrome seen with highly active antiretroviral therapy (HAART), and after rituximab therapy. The exact mechanism responsible for iatrogenic KS flare is unclear. Methods: A case of AIDS-associated cutaneous KS flare following rituximab therapy was compared to similar controls by means of immunohistochemistry using vascular makers (CD34, CD31), monoclonal antibodies to Human Herpesvirus 8 (HHV8) gene products (LNA-1, K5), as well as B-lymphocyte (CD20) and T-lymphocyte (CD3, CD4, CD8) markers. Results: CD20+ B-cell depletion with rituximab in KS flare occurred concomitantly with activation of the HHV8 immediate early gene protein K5. KS flare in this patient was successfully treated with liposomal doxorubicin and valganciclovir. Conclusion: Rituximab-induced KS flare appears to be related to HHV8 activation. Effective management of iatrogenic KS flare therefore depends upon the control of HHV8 viremia in conjunction with specific chemotherapy for KS.

Tiling array-CGH for the assessment of genomic similarities among synchronous unilateral and bilateral invasive breast cancer tumor pairs

2008-07-10

Background: Today, no objective criteria exist to differentiate between individual primary tumors and intra- or intermammary dissemination respectively, in patients diagnosed with two or more synchronous breast cancers. To elucidate whether these tumors most likely arise through clonal expansion, or whether they represent individual primary tumors is of tumor biological interest and may have clinical implications. In this respect, high resolution genomic profiling may provide a more reliable approach than conventional histopathological and tumor biological factors. Methods: 32 K tiling microarray-based comparative genomic hybridization (aCGH) was used to explore the genomic similarities among synchronous unilateral and bilateral invasive breast cancer tumor pairs, and was compared with histopathological and tumor biological parameters. Results: Based on global copy number profiles and unsupervised hierarchical clustering, five of ten (p = 1.9 × 10-5) unilateral tumor pairs displayed similar genomic profiles within the pair, while only one of eight bilateral tumor pairs (p = 0.29) displayed pair-wise genomic similarities. DNA index, histological type and presence of vessel invasion correlated with the genomic analyses. Conclusion: Synchronous unilateral tumor pairs are often genomically similar, while synchronous bilateral tumors most often represent individual primary tumors. However, two independent unilateral primary tumors can develop synchronously and contralateral tumor spread can occur. The presence of an intraductal component is not informative when establishing the independence of two tumors, while vessel invasion, the presence of which was found in clustering tumor pairs but not in tumor pairs that did not cluster together, supports the clustering outcome. Our data suggest that genomically similar unilateral tumor pairs may represent a more aggressive disease that requires the addition of more severe treatment modalities, and underscores the importance of evaluating the clonality of multiple tumors for optimal patient management. In summary, our findings demonstrate the importance of evaluating the properties of both tumors in order to determine the most optimal patient management.

Comparison of DNA histograms by standard flow cytometry and image cytometry on sections in Barrett's adenocarcinoma

Qin Huang, Chenggong Yu, Xiaoqi Zhang and Raj K Goyal — 2008-05-30

Background: The purpose of this study was to compare DNA histograms obtained by standard flow cytometry (FC) and high fidelity image cytometry on sections (ICS) in normal gastrointestinal mucosa and Barrett's adenocarcinoma (BAC). Methods: Archival formalin-fixed paraffin-embedded tissue blocks of 10 normal controls from 10 subjects and 42 BAC tissues from 17 patients were examined. DNA FC was performed using standard techniques and ICS was carried out by Automated Cellular Imaging System (ACIS). DNA ploidy histograms were classified into diploid with peak DNA index (DI) at 0.9–1.1, and aneuploid with peak DI > 1.1. DI values of aneuploid peaks were determined. Additionally, for DNA ICS, heterogeneity index (HI) representing DNA content heterogeneity, and histograms containing cells with DI > G2 were also identified. Results: All control samples were diploid by both FC and ICS analyses. In BAC, FC showed diploid peaks in 29%, diploid peaks with additional aneuploid or tetraploid peaks in 57%, and 14% of the samples, respectively. In contrast, ICS showed aneuploid peaks in all the cases with peak DI > 1.25; 37 cases had peak DI between 1.25 and 2.25; and 5 cases had peak DI > 2.25. HI values (mean ± SD) were 11.3 ± 1.1 in controls and 32.4 ± 8.5 in BAC (p < 0.05). Controls had no G2 exceeding cells. However, 19/37 (51%) of the cases with primary peak DI < 2.25 had cells exceeding 9N. Conclusion: ICS detects DNA aneuploidy in all BAC samples while FC missed the diagnosis of aneuploidy in 29%. In addition, ICS provides more information on HI and G2 exceeding rates.

Population-based study of diagnostic assays for Borrelia infection: comparison of purified flagella antigen assay (Ideia™, Dako Cytomation) and recombinant antigen assay (Liaison®, DiaSorin)

Eskild Petersen, Martin Tolstrup, Francesco Capuano and Svend Ellermann-Eriksen — 2008-04-18

Background: Testing for Borrelia-specific IgM and IgG-antibodies are often performed on a variety of poorly defined symptoms, and isolated IgM results are a frequent finding, which results in diagnostic uncertainty and further testing. We wanted to test the hypothesis that Borrelia-specific assays using recombinant antigens perform differently from assays based on purified flagella antigen. Methods: We compared the use of recombinant antigens (LIAISON® DiaSorin, Saluggia, Italy) and purified flagella antigen (IDEIA™ Borrelia, DakoCytomation, Glostrup, Denmark) in the assay for Borrelia-specific IgM and IgG-antibodies. The assays were tested on an unselected population of serum samples submitted from general practice. A total of 357 consecutive samples for analysis of Borrelia IgM and IgG antibodies. Furthermore, we analysed 540 samples for Borrelia-specific IgM or IgG antibodies first by the IDEIA™ and, if they were positive, the samples were further analysed using the LIAISON® assay. To verify the correctness of the patient's serological status, discrepant samples were analysed by line blots (EcoLine, Virotech). Results: In the consecutive series of 357 samples, the IgM assays detected 308 negative and 3 positive samples with concordant results. Compared with the line blot, the IDEIA™ system produced 21 false-positive IgM results, whereas the LIAISON® system produced only one false-positive IgM result. The IgG assays showed 1 positive and 328 negative concordant results. The LIAISON® system produced 9 true IgG-positive samples that were not detected by the IDEIA™ system, but the former produced 4 positive IgG results that were negative by line blot. Conclusion: Diagnostic assays based on flagella antigen seem to show more false-positive IgM and false-negative IgG results than assays based on recombinant antigens. The latter may reduce the number of presumably false-positive IgM results and identify more IgG-positive subjects, but this system also produces more false-positive IgG results.

Testing for hereditary thrombophilia: a retrospective analysis of testing referred to a national laboratory

Brian R Jackson, Kyland Holmes, Amit Phansalkar and George M Rodgers — 2008-04-02

Background: Predisposition to venous thrombosis may be assessed through testing for defects and/or deficiencies of a number of hereditary factors. There is potential for confusion about which of these tests are appropriate in which settings. At least one set of recommendations has been published to guide such testing, but it is unclear how widely these have been disseminated. Methods: We performed a retrospective analysis of laboratory orders and results at a national referral laboratory to gain insight into physicians' ordering practices, specifically comparing them against the ordering practices recommended by a 2002 College of American Pathologists (CAP) consensus conference on thrombophilia testing. Measurements included absolute and relative ordering volumes and positivity rates from approximately 200,000 thrombophilia tests performed from September 2005 through August 2006 at a national reference laboratory. Quality control data were used to estimate the proportion of samples that may have been affected by anticoagulant therapy. A sample of ordering laboratories was surveyed in order to assess potential measurement bias. Results: Total antigen assays for protein C, protein S and antithrombin were ordered almost as frequently as functional assays for these analytes. The DNA test for factor V Leiden was ordered much more often than the corresponding functional assay. In addition, relative positivity rates coupled with elevations in prothrombin time (PT) in many of these patients suggest that these tests are often ordered in the setting of oral anticoagulant therapy. Conclusion: In this real-world setting, testing for inherited thrombophilia is frequently at odds with the recommendations of the CAP consensus conference. There is a need for wider dissemination of concise thrombophilia testing guidelines.

The calcium-binding protein S100P in normal and malignant human tissues

2008-02-18

Background: S100P is a Ca2+ binding protein overexpressed in a variety of cancers, and thus, has been considered a potential tumor biomarker. Very little has been studied about its normal expression and functions. Methods: We examined S100P expression in normal human tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. S100P protein expression was also studied in a series of tumors, consisting of 74 ovarian, 11 pancreatic, 56 gastric, 57 colorectal, 89 breast and 193 prostate carcinomas using a novel anti-S100P monoclonal antibody. Results: Among the normal tissues, the highest S100P mRNA levels were observed in the placenta and esophagus. Moderate signals were also detected in the stomach, duodenum, large intestine, prostate and leukocytes. At the protein level, the highest reactions for S100P were seen in the placenta and stomach. Immunostaining of tumor specimens showed that S100P protein is expressed in all the tumor categories included in the study, being most prevalent in gastric tumors. Conclusion: Based on our observations, S100P is widely expressed in both normal and malignant tissues. The high expression in some tumors suggests that it may represent a potential target molecule for future diagnostic and therapeutic applications.

Preservation of biomolecules in breast cancer tissue by a formalin-free histology system

2008-01-29

Background: The potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer – estrogen receptor and HER2 – at the RNA and protein levels. Methods: Parallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fixative and in formalin. Molecular fixative samples were processed by a novel formalin-free microwave-assisted processing system that preserves DNA, RNA and proteins. Formalin-fixed samples were processed using the conventional method. Estrogen receptor was assessed by immunohistochemistry and real-time PCR. HER2 was assessed by immunohistochemistry, FISH, CISH and real-time PCR. Results: The immunohistochemical reaction for estrogen receptor was similar in molecular- and formalin-fixed samples (Spearman Rank R = 0.83, p < 0.05). Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05). The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative. Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens. Conclusion: The formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.

Biological variability dominates and influences analytical variance in HPLC-ECD studies of the human plasma metabolome

2007-11-12

Background: Biomarker-based assessments of biological samples are widespread in clinical, pre-clinical, and epidemiological investigations. We previously developed serum metabolomic profiles assessed by HPLC-separations coupled with coulometric array detection that can accurately identify ad libitum fed and caloric-restricted rats. These profiles are being adapted for human epidemiology studies, given the importance of energy balance in human disease. Methods: Human plasma samples were biochemically analyzed using HPLC separations coupled with coulometric electrode array detection. Results: We identified these markers/metabolites in human plasma, and then used them to determine which human samples represent blinded duplicates with 100% accuracy (N = 30 of 30). At least 47 of 61 metabolites tested were sufficiently stable for use even after 48 hours of exposure to shipping conditions. Stability of some metabolites differed between individuals (N = 10 at 0, 24, and 48 hours), suggesting the influence of some biological factors on parameters normally considered as analytical. Conclusion: Overall analytical precision (mean median CV, ~9%) and total between-person variation (median CV, ~50–70%) appear well suited to enable use of metabolomics markers in human clinical trials and epidemiological studies, including studies of the effect of caloric intake and balance on long-term cancer risk.

Low specificity of HIV-testing on sputum specimens kept at ambient temperatures for 4 to 7 days: a blinded comparison

2007-09-19

Background: HIV testing on sputum using the QraQuick HIV1/2® assay has high sensitivity and specificity, and holds promise for application in tuberculosis surveys. Its performance under conditions that may occur during surveys in resource-poor countries is however, unknown. We assessed, in a blinded comparison with HIV serum testing, the sensitivity and specificity of the OraQuick® assay for detecting HIV antibody in sputum specimens kept at ambient temperature for up to 7 days, with and without decontaminant. Methods: Paired sputum and blood specimens from consecutively diagnosed smear-positive tuberculosis patients were tested with OraQuick® and 2 HIV-1/2 ELISA's. Sputum was tested within 24 hours of collection, split into 2 aliquots with and without addition of cetylpyridium chloride, and tested again after 4 and 7 days. Results: Complete data was available for 377/435 (87%) enrolled patients; 132 (35%) tested HIV positive on serum. The sensitivity of the sputum test was 94.7% (95% CI 89.4–97.8) on day 1, 93.2% on day 4 and 92.9% on day 7. The specificity was 92.9% (95% CI 88.9–95.8) on day 1, and declined to 76.7% on day 4 (p < 0.001) and to 62.7% on day 7 (p < 0.001). Adding cetylpyridium chloride further decreased the specificity to 67.8% on day 4 (p = 0.04) and to 49.6% on day 7 (p = 0.004). Conclusion: Transportation of sputum specimens at ambient temperatures for 4 days or more, and addition of decontaminant, strongly affect the specificity of the OraQuick® assay. Unless applied within one day, this assay is not suitable for estimation of HIV-prevalence among tuberculosis patients in survey settings.

An exfoliation and enrichment strategy results in improved transcriptional profiles when compared to matched formalin fixed samples

Wilfrido D Mojica, Leighton Stein and Lesleyann Hawthorn — 2007-08-03

Background: Identifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material. Methods: Triplicate samples of non-neoplastic colonic epithelial cells were recovered from a hemicolectomy specimen using three different procurement methods from the same originating site: 1) an exfoliation and enrichment strategy 2) laser capture microdissection from formalin fixed tissue and 3) laser capture microdissection from frozen tissue. Parameters currently in use to assess RNA integrity were utilized to assess the quality of recovered RNA. Additionally, an expression microarray was performed on each sample to assess the influence each procurement technique had on RNA recovery and degradation. Results: The exfoliation/enrichment strategy was quantitatively and qualitatively superior to tissue that was formalin fixed. Fixation negatively influenced the expression profile of the formalin fixed group compared to both the frozen and exfoliated/enrichment groups. Conclusion: The exfoliation/enrichment technique represents a superior alternative in tissue procurement and RNA recovery relative to formalin fixed tissue. None of the deleterious effects associated with formalin fixation are encountered in the exfoliated/enriched samples because of the absence of its use in this protocol. The exfoliation/enrichment technique also represents an economical alternative that will yield comparable results to cells enriched by laser capture microdissection from frozen tissue sections.