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Quantitation of chromosome 1p and 19q deletions in glial tumours by interphase FISH on formalin-fixed paraffin-embedded tissue.

Deletions on chromosomes 1p and 19q identify oligodendroglial tumours that are likely to have a complete response to some chemotherapy regimens and are associated with prolonged patient survival. Detection of these chromosomal alterations is becoming increasingly important in the evaluation of glial tumours. Interphase fluorescence in situ hybridisation (FISH) is one of a number of techniques for detecting deletions, and is an efficient method for screening large numbers of tumours. We used FISH to detect 1p and 19q deletions in formalin-fixed paraffin sections of 11 oligodendrogliomas, 11 oligoastrocytomas, two astrocytomas and four glioblastomas multiforme. 1p and 19q deletion and non-deletion ratios were tabulated in 200 nuclei in each tumour. We found considerable variation between tumours in the fraction of cells with deletions. This variation has not been reported previously and its clinical significance will be clarified with patient follow-up. There was little variation between regions within the same tumour.

Brain Neoplasms↗

A new proliferation-associated nuclear antigen detectable in paraffin-embedded tissues by the monoclonal antibody Ki-S1.

A monoclonal antibody (Ki-S1) has been raised that reacts with the nuclei of proliferating cells. The antigen recognized is resistant to formalin fixation and can be detected in frozen tissues as well as in routinely processed specimens. In immunohistochemistry, nuclear staining can be seen in those tissues and cellular compartments known to be actively proliferating. Peripheral blood lymphocytes are negative but show a strong increase in antigen expression after mitogen stimulation. Flow cytometric determination of DNA content and antigen expression revealed negativity of G0 cells and positivity of G1 to G2/M cells. A cytoplasmic co-reactivity, not associated with proliferation, was confined to Langerhans islands of the pancreas. The nuclear localized antigen has a molecular mass of 160 kd and therefore seems to be different from all other known immunohistochemical markers of proliferating cells. We conclude that the monoclonal antibody Ki-S1 might provide a useful tool for studying cell proliferation in situ under normal and pathological circumstances.

Animals↗

Inter-laboratory validation of PCR-based detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues.

The present study is based on the initiative for quality assurance in pathology of the German Society of Pathology and the Professional Association of German Pathologists. Four panel laboratories with experience and expertise in polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis were selected to establish the prerequisites for continuous external laboratory trials, in particular, by providing pre-tested specimens and evaluation criteria for participating institutes. In the first step, the four panel laboratories performed an internal trial to test their own reliability and reproducibility. Paraffin sections and DNA preparations from 34 tissues (25 clinical specimens and 9 controls) totalling to 66 samples were evaluated by each panel institute according to their own protocols. The methodologies differed and are described in detail. Despite these differences, a high degree of inter-laboratory reliability was achieved. In this report, we summarise our results including the correlation with the histology and provide recommendations for applying PCR-based methodology for the detection of mycobacterial DNA in surgical specimens. Supplementary data are available online at http://www.charite.de/ch/patho (rubric "Forschung"). Pre-tested specimens are now available for the external trial and can be ordered from the steering institute via Oligene (http://www.oligene.com/). All molecular pathology laboratories are invited to participate in this quality assurance initiative.

Animals↗

Three-dimensional reconstruction of tumor microvasculature: simultaneous visualization of multiple components in paraffin-embedded tissue.

Three-dimensional (3D) visualization of microscopic structures may provide useful information about the exact 3D configuration, and offers a useful tool to examine the spatial relationship between different components in tissues. A promising field for 3D investigation is the microvascular architecture in normal and pathological tissue, especially because pathological angiogenesis plays a key role in tumor growth and metastasis formation. This paper describes an improved method for 3D reconstruction of microvessels and other microscopic structures in transmitted light microscopy. Serial tissue sections were stained for the endothelial marker CD34 to highlight microvessels and corresponding images were selected and aligned. Alignment of stored images was further improved by automated non-rigid image registration, and automated segmentation of microvessels was performed. Using this technique, 3D reconstructions were produced of the vasculature of the normal brain. Also, to illustrate the complexity of tumor vasculature, 3D reconstructions of two brain tumors were performed: a hemangioblastoma and a glioblastoma multiforme. The possibility of multiple component visualization was shown in a 3D reconstruction of endothelium and pericytes of normal cerebellar cortex and a hemangioblastoma using alternate staining for CD34 and alpha-smooth muscle actin in serial sections, and of a GBM using immunohistochemical double staining. In conclusion, the described 3D reconstruction procedure provides a promising tool for simultaneous visualization of microscopic structures.

Brain Neoplasms↗

Desmoplastic small round-cell tumor of the paratesticular region: report of an adult case with demonstration of EWS and WT1 gene fusion using paraffin-embedded tissue.

Desmoplastic small round-cell tumor typically occurs in the abdomen of young men, but it can also develop at other anatomic sites and in older people, presenting greater diagnostic difficulties. We report a case of this tumor arising from the paratesticular region in a 43-year-old man. The tumor showed histologic, immunohistochemical, and ultrastructural evidence of multilineage differentiation, including epithelial, mesenchymal, and neuronal features. In addition, the presence of an EWS and WT1 chimeric messenger RNA was demonstrated by the reverse transcriptase-polymerase chain reaction using an EWS exon 7 primer and WT1 exon 8 and exon 9 primers, which revealed single polymerase chain reaction products with a junction of EWS exon 7 to WT1 exon 8. Our study demonstrates that desmoplastic small round-cell tumors of the paratesticular region share not only morphologic but also molecular genetic features with those of the abdomen and that reverse transcriptase-polymerase chain reaction analysis using paraffin sections is useful for a conclusive diagnosis.

Abdominal Neoplasms↗

Microsatellite instability is rare in the clinical course of myelodysplastic syndrome studied with DNA from fresh and paraffin-embedded tissues.

Microsatellite instability (MSI) has been reported to occur in various types of malignant neoplasms. We performed a polymerase-chain-reaction-based assay for MSI between the initial and the most recently available ("latest") samples from 23 patients with myelodysplastic syndrome (MDS). Of these patients, 15 were informative at more than three microsatellite loci. Seven patients showed an increase in leukemic cells while 8 patients did not during the interval between the two analyses. Only 1 of the patients, who had refractory anemia with excess blasts, which changed to acute myelogenous leukemia, showed microsatellite alteration at the analysis times. Among all 23 patients, two alterations were detected in the 42 informative paired samples that showed an increase in leukemic cells (4.8%), while none was detected in the 59 paired samples without such an increase. In total, therefore only two alterations were detected among 101 informative paired samples (2%). This indicates that MSI is rare in the clinical course of MDS irrespective of disease status, and is consequently not a critical genetic event for disease progression in most MDS patients.

Adult↗

Diagnostic tools for differentiating pleural mesothelioma from lung adenocarcinoma in paraffin embedded tissue. II. Design of an expert system and its application to the diagnosis of mesothelioma.

A panel of 14 antibodies (panepithelial antibody Lu-5, anti-keratin-18, anti-keratin-7, Ber-EP4, anti-Leu-M1, HEA-125, anti-carcinoembryonic antigen, anti-blood group-related antigens A, B, H, B72.3, anti-placental alkaline phosphatase, anti-vimentin and BMA-120), which have been evaluated for use in differentiating mesothelioma from lung adenocarcinoma, was applied to a group of 24 suspected mesotheliomas. Using the established qualitative, descriptive criteria derived from monovariate statistical analysis of the tumour control groups (definite mesotheliomas, adenocarcinomas), a definitive allocation was possible in only 25% of suspected cases. We therefore constructed two "expert systems", based on multivariate discriminant analysis with either the ALLOC 80 program for ordinal data or a newly developed analysis program for binomial data. With these two systems diagnostic allocation of suspected mesotheliomas was improved to 75% and 79%. The use of binomial data ("positive" versus "negative") in conjunction with the probability-based test system is of particular interest because the primary data are easy to record and the test results have a higher statistical probability.

Adenocarcinoma↗

Grading melanocytic dysplasia in paraffin wax embedded tissue by the nucleic acid index.

BACKGROUND: Although nucleic acid derangements are the hallmark of melanocytic dysplasia, the gold standard for its diagnosis remains the microscopic evaluation of haematoxylin and eosin stained slides. However, light microscopy is subjective and crucial genomic changes do not always show as changes in histology. AIMS: To introduce the nucleic acid index (NAI) as a means of analysing nucleic acid derangements in histological sections at the level of the individual cell and within the context of its microenvironment. METHODS: Confocal laser scanning microscopy was performed on melanocytic lesions stained with acridine orange (AO), a fluorescent stain for DNA and RNA. The NAI, calculated by measuring the fluorescence intensities of AO in nuclei relative to the surrounding cytoplasm, reflects the concentration of DNA relative to RNA. RESULTS: When applied to benign naevi, dysplastic naevi, and melanoma, a very strong significant association was seen between lower NAI and malignant potential (p < 0.0001). Strong inverse correlations were found between NAI and both mitotic index and Breslow thickness. Interestingly, the NAI for dysplastic naevi is between that of melanoma and most benign naevi, consistent with their intermediate biological behaviour and histological appearance. CONCLUSION: By providing a quantitative measure for melanocytic neoplasia, the NAI may improve the diagnosis of melanocytic lesions and the selection of treatment.

DNA, Neoplasm↗

Immunohistochemical studies on c-erbB-2 oncoprotein expression in paraffin embedded tissues in invasive and non-invasive human breast lesions.

The c-erb-B2 protein expression was evaluated by immunohistochemistry in two series of breast lesions. In a retrospective study on a series of 140 breast lesions, among 34 benign lesions, 16 showed an intense or moderate membrane reaction of tumoral cells; of 15 atypical lesions, 6 exhibited a membrane reaction and 34/60 in situ lesions expressed intensely or moderately c-erb-B2 oncoprotein. A prospective investigation was made on a series of 41 lesions from 25 patients, which were selected for their clinico-pathological features. All the samples were positive but the staining intensity was heterogenous. However, it was similar in the various lesions from the same patient. A strong signal was observed in 12 lesions from 7/25 patients, moderate in those of 13 patients and weak in 6 lesions from 5 patients. Taken together, our findings show that a large number of human breast lesions expressed c-erb-B2 oncoprotein, whether benign, atypical or in situ carcinomas. The intensity of the reaction may depend on the patients rather than on the aggressivity of the lesion, except for the infiltrating carcinomas. This suggests that c-erb-B2 oncoprotein in overexpressed at early stages of breast cancer lesions. Although c-erb-B2 oncoprotein is probably not a good marker for prognosis, it may be an indication for a future progression towards or recurrence.

Adult↗

Common ALK gene rearrangement in Asian CD30+ anaplastic large cell lymphoma: an immunohistochemical and fluorescence in situ hybridisation (FISH) study on paraffin-embedded tissue.

AIMS: The most common recurrent genetic aberration in anaplastic large cell lymphoma (ALCL) is translocation involving the ALK gene that results in ectopic expression of ALK protein in lymphoid tissue. This study aims to investigate the frequency of ALK gene rearrangement in a series of Asian ALCL. METHODS: ALK gene rearrangement was detected by immunostaining of ALK protein and fluorescence in situ hybridisation (FISH) targeting at the 2p23 region. RESULTS: The expression of ALK protein was detected in 24/34 (71%) of the cases, and it was significantly higher in childhood cases (100%) when compared to adult cases (47%). The analyses by FISH were consistent with the results from immunostaining of ALK protein, but the analyses were only successful in 15/34 (44%) cases. FISH analyses detected extra copies of ALK gene in three cases, including one case that expressed ALK protein and showed 2p23 rearrangement. CONCLUSIONS: The current series revealed a high frequency of ALK gene rearrangement, especially in the children. Immunostaining of ALK protein is a reliable indication of ALK gene rearrangement, and is superior to FISH. However, FISH analysis is useful in detecting other genetic aberrations that are not related to ALK gene rearrangement.

Adolescent↗

Fluorescence in situ hybridization analysis of chromosome 12p in paraffin-embedded tissue is useful for establishing germ cell origin of metastatic tumors.

The over-representation of chromosome 12p sequences is crucial for the development of invasive testicular germ cell tumors. Testicular cancer patients may have metastatic tumors of diverse histologic types, including adenocarcinoma, undifferentiated carcinoma, sarcoma, or other malignancies that lack features of germ cell tumors. We sought to investigate the possible germ cell origin of such tumors using interphase fluorescence in situ hybridization. In all, 10 metastatic malignant somatic-type tumors from patients with histories of testicular cancer, as well as one malignant somatic-type tumor from a patient with primary mediastinal germ cell tumor were studied and included: adenocarcinoma (five cases), poorly differentiated carcinoma (one), sarcoma (four), and neuroendocrine carcinoma (one). The tumors were analyzed using fluorescence in situ hybridization using 12p spectrum green and 12 centromeric spectrum orange probes in paraffin sections. The patients ranged in age from 27 to 55 years (mean, 43). Colon and lung cancers from patients without germ cell tumors were used as controls. Adequate signals were observed in all tumors. Gain of chromosome 12p was seen in six tumors. None of the control tumors showed 12p amplification. Fluorescence in situ hybridization for 12p amplification in routinely processed surgical specimens is a useful adjuvant diagnostic tool in confirming the germ cell origin of metastatic tumors having the histologic appearance of somatic-type neoplasms.

Adenocarcinoma↗

Evaluation of diagnostic efficiency of computerized image analysis based quantitative nuclear parameters in papillary and follicular thyroid tumors using paraffin-embedded tissue sections.

Computerized image analysis (IA) system has emerged in recent years as a very powerful tool for objective and reproducible quantification of histological features. It has shown considerable potential for diagnostic application in diverse histological situations. The objectives of the present study were to evaluate the discriminatory diagnostic efficiency of computerized image analysis based quantitative subvisual nuclear parameters in papillary and follicular neoplasms of thyroid. A total of 60 cases were studied. Forty-four cases belonged to training set and 16 cases belonged to a test set. A minimum of 100 nuclei was analyzed in each case using uniform 5 m mm thick hematoxylin stained sections. The IA workstation comprised of an Olympus microscope, a 10 bit digital video camera, an image grabber card and a pentium 120 MHz computer. Optimas 5.2 software was utilized for data collection on 8 morphometric and 8 densitometric parameters. Multivariate stepwise discriminant statistical analysis of data was done with the help of BMDP statistical software release 7.0. Results from a training set revealed correct classification rates of 98.0%, 84.5% and 61.2% for the histological groups of hyperplastic papillae versus papillae of papillary carcinoma (group I), follicular variant of papillary carcinoma versus the broad category of follicular neoplasms consisting of both follicular adenoma and follicular carcinoma (group II) and follicular adenoma versus follicular carcinoma (group III), respectively. Results of test set revealed correct classification rates of 100%, 80% and 50% for groups I, II and III respectively. It was concluded that computerized nuclear IA parameters have potential usefulness for discriminating benign versus malignant papillary lesions of thyroid, follicular variant of papillary carcinoma versus follicular adenoma and/or follicular carcinoma but are of no value in discriminating between follicular adenoma and follicular carcinoma.

Adenocarcinoma, Follicular↗

DNA flow cytometry of fresh and paraffin-embedded tissue using cytokeratin staining.

DNA flow cytometry measurements were performed using cytokeratin as a second parameter to identify epithelial cells selectively in fresh and in archival paraffin samples of normal and adenocarcinoma tissues from breast and colon. Fresh specimens consisted of 22 adenocarcinomas of breast, 20 adenocarcinomas of colon, 16 control breast samples, and 13 control colon samples. Paraffin block specimens consisted of 22 adenocarcinomas of breast (the same as fresh samples), 20 adenocarcinomas of colon (the same as fresh samples), 37 control breast samples and 34 control colon samples. The average proportion of cytokeratin-positive cells per group ranged from 31 to 55% for fresh samples and from 14 to 34% for paraffin samples. For aneuploid cells populations of adenocarcinomas, which consist only of epithelial cells, the average percentage of cytokeratin-positive cells ranged from 60 to 72%. The technique gave satisfactory measurements of ploidy and of cell cycle data in both types of samples. Cell cycle measurements were less accurate than ploidy measurements in both types of samples, and multiple sampling will be required for adequate accuracy. The average S-phase fraction of cytokeratin-positive cells ranged from 6 to 15% for fresh specimens and from 11 to 20% for paraffin samples. Similar data were obtained for the proliferative index (G1 + S + G2 + M phases). The coefficients of variation were smaller for proliferative index than for S-phase fraction data, indicating greater accuracy. Paraffin data give higher cycling cell measurements than corresponding fresh data, so separate standardization of measurements may be required for fresh and for paraffin data.

Adenocarcinoma↗

Low CD7 expression in benign and malignant cutaneous lymphocytic infiltrates: experience with an antibody reactive with paraffin-embedded tissue.

Loss of CD7 expression by neoplastic lymphocytes is considered a distinguishing characteristic of mycosis fungoides (MF) and cutaneous T-cell lymphoma. Reports to date examining for the CD7 immunophenotype in MF have been performed on fresh-frozen tissue. In this study, we used a paraffin-reactive antibody directed against CD7 to determine its range of expression in MF and to compare these results with those in controls. Examining 22 cases of MF and 61 controls, we found minimal CD7 expression by lymphocytes in MF and in a few cases of benign inflammatory dermatosis (BID). The lowest mean CD7 counts (as a percentage of total lymphocytes) were found in MF (patch stage: 5% +/- 5%, range: 0-10; plaque and tumor stages: 15% +/- 5%, range: 5-25), and these counts were significantly lower than those for BID (35% +/- 20%, range: 5-80; p = 0.001). By logistic regression analysis, low CD7 expression (<10% lymphocytes labeling) had sensitivity and positive predictive values of 80% and 72%, respectively, and specificity and negative predictive values of 93% and 96%, respectively, for the diagnosis of patch stage MF. False-positive results were found for spongiotic dermatitis. Moreover, spongiotic dermatitides exhibited a progressive decrease in mean CD7 counts from acute to subacute to chronic stages (50% versus 35% versus 30%, respectively). In conclusion, minimal CD7 expression is a specific finding for MF. Benign inflammatory infiltrates can also show low CD7 expression, however, which rarely matches that of patch stage MF. Progressive loss of CD7 expression in BID is the likely consequence of expansion of antigen-selected CD3+CD4+CD7- T cells. These inflammatory CD4+CD7- T cells may represent the physiologic counterpart to the neoplastic lymphocyte of MF.

Antibodies, Neoplasm↗

Rapid automated combined in situ hybridization and immunohistochemistry for sensitive detection of cytomegalovirus in paraffin-embedded tissue biopsies.

The authors questioned whether an automated kinetic mode assay of combined cytomegalovirus (CMV) late viral message and immediate and early antigens might result in a more sensitive and timely CMV diagnosis relevant to speedy treatment in the transplant setting. Toward this end, two cohorts were studied using automated in situ hybridization (ISH) for CMV as well as immunohistochemistry (IHC). The first cohort of patients consisted of 19 cases that were histologically positive (CMV-associated cytopathic change). A second cohort consisted of 10 cases that were histologically negative, yet culture positive. From the first cohort of histologically positive cases, 100% were positive by both ISH and IHC run on separate slides. In the second cohort, CMV was detected overall in 70% of cases (50% by ISH alone and 30% by IHC alone). These results indicate that a combined assay of ISH and IHC can detect more cases than routine hematoxylin and eosin staining or either assay alone. In two illustrative cases, used to demonstrate the feasibility of combining ISH and IHC, the authors used a combined two-color assay (ISH and IHC) performed sequentially on the same slide. The combined assays resulted in colocalized single cell message and protein in some cells and demonstrated more positive cells overall (some positive by IHC alone, some by ISH alone, and some by both) than either assay alone. The combined dual color assay can be completed within 4 to 5 hours giving the prospect of a same day result, which is faster than shell vial technique with immunofluorescence (24 to 48 hours) or culture (7 to 14 days). This study demonstrates that combining CMV message and protein assays results in a more sensitive assay and, when carried out in the kinetic mode, allows a speedy result relevant to early anti-CMV therapy.

Aged↗

Usefulness of an immunohistochemical panel in paraffin-embedded tissues for the differentiation of B-cell non-Hodgkin's lymphomas of small lymphocytes.

Diagnosis of small B-cell lymphomas is sometimes difficult without fresh tissue for flow cytometry (FC) or immunohistochemistry (IHC). Therefore, we examined the usefulness of a paraffin section IHC panel consisting of antibodies to CD5, CD10, CD20, CD23, CD43, and cyclin D1. We tested 55 formalin-fixed small B-cell lymphomas, including 16 small lymphocytic lymphomas (SLLs), 10 mantle cell lymphomas (MCLs), 25 follicle center lymphomas (FCLs), and 4 mantle zone lymphomas (MZLs). Seventeen cases had B5-fixed sections that were stained in the same manner. The findings were correlated with FC immunophenotyping when available. All of the SLLs and 90% of the MCLs expressed CD5 by IHC, with occasional weak expression in some MCLs. All of the FCLs and MZLs lacked CD5 expression. These results were comparable to those obtained by FC. CD43 expression was seen in 100% of the SLLs, 90% of the MCLs, and 75% of the MZLs. CD23 expression was seen in 94% of the SLL; of these, 100% also showed expression of CD23 by FC. Cyclin D1 was detected in all of the MCLs by IHC but also in 3 of the 16 SLLs. CD23 was absent in all of the MCLs. CD10 expression was present in 21 (95%) of 22 FCLs. All of the 17 cases fixed in B5 showed a decreased immunoreactivity for CD5 in the neoplastic cells. In contrast, CD10 immunoreactivity was judged better in B5-fixed sections. We concluded, therefore, that anti-CD5 and -CD10 were useful tools in the differential diagnosis of B-cell lymphomas of small lymphocytes and that a paraffin-section IHC panel consisting of antibodies to CD5, CD10, CD20, CD23, CD43, and cyclin D1 was a useful ancillary technique that compared favorably with FC.

Antigens, CD↗

Sexing of human embryos and fetuses by fluorescent in situ hybridization (FISH) to paraffin-embedded tissues with sex chromosome-specific DNA probes.

A fluorescent in situ hybridization (FISH) study was carried out on paraffin sections of human embryonic and fetal tissues with two DNA probes, DXZ1 and DYZ1 (Oncor), for X and Y chromosome-specific DNA sequences, respectively. The specificity of the DNA probes was confirmed on metaphase and interphase lymphocytes of healthy normal adult males. The paraffin blocks of the human embryonic and fetal tissues examined in the present study had been stored at room temperature for up to 5 years after fixation in 4% paraformaldehyde. All the seven embryos and five fetuses examined were successfully sexed by FISH. The cells from three embryos and four fetuses were positive for a hybridization signal with each of the DXZ1 and DYZ1 probes and they were classified as male. The cells from the remaining four embryos and one fetus were positive for two identical hybridization signals with the DXZ1 probe in a nucleus instead of the absence of the signal hybridized with DYZ1, indicating that their cells have two X chromosomes but no Y chromosomes. The FISH results for the five fetuses examined were consistent with their genital sex and/or gonadal histology. The FISH results were highly specific and no false positive or false negative results were obtained. Thus, the FISH technique has been shown to visualize specific DNAs in situ on paraffin sections and to be useful to determine the sex of fixed embryos and fetuses retrospectively.

Cells, Cultured↗