Hypothermic circulatory arrest with "low flow" lower body perfusion: an experimental feasibility study of microcirculatory parameters.

BACKGROUND: To avoid extended cardiopulmonary bypass (CPB), moderate temperatures are commonly accepted for hypothermic circulatory arrest (HCA), thereby jeopardizing organ protection. Distal aortic perfusion may be an option, but supportive experimental data is missing. METHODS: Eight juvenile pigs (36 ± 2 kg) were cooled to 30 °C followed by 60 min of HCA with 50 min of low flow (LF) lower body perfusion. Multimodal monitoring was used to measure overall metabolism, hemodynamics and microcirculation of the terminal ileum. The animals were observed for four hours following reperfusion. Organs were harvested for histopathological evaluation. RESULTS: During LF perfusion, initially elevated l-lactate levels decreased subsequently ( P < 0.05). Capillary blood flow decreased during cooling to 50 % baseline levels ( P = 0.03), but remained stable under LF conditions. Parameters indicative of reduced liver and kidney function were slightly elevated at the end of the experiment, but still within normal ranges. CONCLUSION: Under moderate hypothermia, low flow perfusion seems to provide adequate protection for the lower body organs. Microcirculatory parameters during perfusion as well as lactate levels within normal ranges throughout the experiments further confirm the concept.

Primary cervical cancer truly negative for high-risk human papillomavirus is a rare but distinct entity that can affect virgins and young adolescents.

Cancer of the uterine cervix is almost exclusively associated with human papillomavirus (HPV). Carcinogenesis is slow, the minimal time from initial HPV infection to invasive carcinoma seems to be less than ten years. In order to identify rapid onset cervical cancer, we carried out a retrospective re-analysis of an extended cohort of patients with invasive cervical cancer, and reviewed cases identified within the cancer registry of Lower Saxony or using Medline or ISI data. No instances of a rapid-onset cancer or true HPV-DNA negative cancer were found among our hospital cohort of 178 women with primary cancer of the uterine cervix. Registry data identified four out of 5,878 patients who were diagnosed with primary cervical cancer at 14 to 20 years of age. They were classified as clear-cell and endometriod adenocarcinoma and tested persistently negative for high-risk HPV-DNA. Fourteen more cases of cervical cancer in virgins and very young women were identified by a Medline search, mostly with unknown histologic type or rare subtypes of adenocarcinoma. In conclusion, rare adenocarcinoma of the uterine cervix may represent an entity unrelated to HPV, thus explaining instances of rapid onset cervical cancer.

[Interlaboratory trials for quality assurance of breast cancer biomarkers in Germany]. / Ringversuche zum Nachweis von therapeutischen Zielmolekülen beim Mammakarzinom in Deutschland.

In the age of personalized medicine, and in addition to typing and grading, breast cancer pathologists are now also involved in determining biomarkers such as steroid hormone receptors and Her-2, which are of the utmost importance in adjuvant therapy. In order to assure quality of these biomarker assays, external proficiency testing has been implemented in Germany. Since 2002 trials have been conducted annually, with up to 180 participating laboratories. More than 85% of all participants achieved good results in clearly negative and positive cases seen in daily practice. If at all, discordant results were observed in the rarer low steroid-hormone receptor expressing tumors and Her-2 borderline cases (2+). Regular participation in interlaboratory testing leads to significantly improved immunohistochemical results, particularly in these problematic cases. Tissue microarrays (TMA) with 20-24 different breast cancer samples including cell lines meant that a huge number of pathologists were challenged with identical samples, providing the prerequisite for comparability. Participation is recommended for pathology departments involved in the service for breast units. The organizational frame work of the trials is described here. The confidence of cooperating disciplines in breast cancer biomarkers assessed by pathologists will be fostered by external proficiency testing as presented here.

Immunocytological detection of lymph node metastases in dogs with malignant epithelial tumours.

The aim of this study was to compare immunolabelling of cytological specimens with conventional staining in the detection of metastases in lymph nodes from dogs with carcinoma. Cytological touch imprints of 161 lymph nodes from 72 dogs, as well as 50 fine needle aspirates from 23 dogs, with malignant epithelial tumours were included in the study. Immunolabelling was performed with commercially available human antibodies. Touch imprints of all lymph nodes were labelled with broad spectrum anticytokeratins AE1/AE3 and KL1. In addition, lymph node touch imprints from dogs with primary tumours that reacted positively with the specific anticytokeratins CK7 (n=104) and CK20 (n=20) were also labelled with CK7 and CK20. Fine needle aspirates of 50 lymph nodes were examined by immunolabelling with AE1/AE3. "Reference investigations" with a combination of histological and immunohistochemical methods were performed on all lymph nodes. The immunocytological detection of lymph node metastases with the broad spectrum anti-cytokeratin AE1/AE3 in imprint smears resulted in a significant increase in sensitivity (0.99 vs 0.88 [conventional stain]) and in negative predictive value (0.99 vs 0.85) (P<0.01; t-test). Micrometastases in particular were detected more readily. Specificity (0.93 vs 0.88) and positive predictive value (0.95 vs 0.90) did not differ significantly between the two techniques. Immunolabelling with KL1 was associated with lower sensitivity and negative predictive value, indicating lack of cross-reactivity of this antibody with canine epithelial cells. In fine needle aspirates the detection of lymph node metastases, especially micrometastases, was more efficient by mean of immunolabelling with AE1/AE3 than by conventional staining. The study indicated the value of immunocytological labelling for the detection of metastases in cytological specimens of canine lymph node preparations.

Quality assurance trials for Ki67 assessment in pathology.

Ki67 is a broadly used proliferation marker in surgical pathology with an obvious need for standardization to improve reproducibility of assessment. Here, we present results of the so far only existing round robin tests on Ki67, organized annually in Germany, Austria, and Switzerland from 2010 to 2015 with up to 160 participating laboratories (QuIP). In each quality assessment trial, eight probes from each breast cancer, neuroendocrine tumor, and malignant lymphoma were compiled on a tissue microarray (TMA). TMAs were stained in the participants' laboratories with antibodies and procedures also applied in their daily routine. Participating pathologists were expected to assign Ki67 values to one of four different categories for each tumor type. All local stainings and evaluations were reassessed by the organizing panel and compared to a preset standard. On average, 95% of participants reached the benchmark of over 80% concordance rates with the Ki67 category pre-established by the panel. Automatization and type of antibody did not affect the success rate. Concordance rates differed between tumor entities being highest in each tumor type with either very high or very low labeling indices. Lower rates were seen for intermediate Ki67 levels. Staining quality improved during the observation period as did inter-observer concordance with 85% of participants achieving excellent agreement (kappa > 0.8) in the first year and over 95% in 2015. In conclusion, regular external quality assurance trials have been established as a tool to improve the reproducibility and reliability of the prognostic and predictive proliferation marker Ki67.

Wnt-5a has tumor suppressor activity in thyroid carcinoma.

Stabilization of beta-catenin by inhibition of its phosphorylation is characteristic of an activation of the canonical Wnt/beta-catenin signaling pathway and is associated with various human carcinomas. It contrasts to an as yet incompletely characterized action of an alternative noncanonical Wnt signaling pathway on neoplastic transformation. The aim of the present study was to test the effects of a member of the noncanonical Wnt signaling pathway, Wnt-5a, in primary thyroid carcinomas and in thyroid carcinoma cell lines. Compared to normal tissue Wnt-5a mRNA expression was clearly increased in thyroid carcinomas. Immunohistochemically, a bell-shaped response was observed with low to undetectable levels in normal tissue and in anaplastic tumors whereas differentiated thyroid carcinomas showed strong positive immunostaining for Wnt-5a. Transfection of Wnt-5a in a thyroid tumor cell line FTC-133 was able to reduce proliferation, migration, invasiveness and clonogenicity in these cells. These effects of Wnt-5a are associated with membranous beta-catenin translocation and c-myc oncogene suppression and are mediated through an increase in intracellular Ca(2+) release, which via CaMKII pathways promotes beta-catenin phosphorylation. Specific inhibition of beta-catenin phosphorylation by W-7, a calmodulin inhibitor, or by KN-93, a CaMKII inhibitor, supports these findings whereas PKC inhibitors were without effect. This interaction occurs downstream of GSK-3 beta as no Wnt-5a effect was seen on the Ser(9) phosphorylation of GSK-3 beta. Our data are compatible with the hypothesis that Wnt-5a serves as an antagonist to the canonical Wnt-signaling pathway with tumor suppressor activity in differentiated thyroid carcinomas.

Pitfalls in immunohistochemical assessment of EGFR expression in soft tissue sarcomas.

BACKGROUND: New targeted cancer treatments acting against growth factor receptors such as the epidermal growth factor receptor (EGFR) necessitate selecting patients for treatment with these drugs. Besides carcinomas, soft tissue sarcomas (STS) express EGFR and might thereby be a promising target for this new therapeutic strategy. OBJECTIVE: To test and compare different EGFR antibodies to determine the frequency of EGFR expression in STS. METHODS: 302 consecutive specimens of STS were examined using the tissue microarray technique. EGFR expression levels were assessed by immunohistochemistry using five different commercially available antibodies. Gene amplification status was measured by fluorescence in situ hybridisation (FISH). Immunoreactivity and amplification status were correlated with clinicopathological features and follow up data available in 163 cases. RESULTS: EGFR expression frequency ranged between 0.3% and 52.9%, depending on the antibody and scoring method used. In all, 3.5% of the tumours showed egfr gene amplification by FISH, which correlated with EGFR expression for three antibodies. Only one antibody had independent prognostic value in multivariate analysis and correlated with an unfavourable outcome; egfr gene amplification status showed no correlation with clinical features. CONCLUSIONS: Frequency of EGFR immunopositivity in STS strongly depends on the antibody used, and only one of five antibodies tested predicted an unfavourable clinical outcome. This indicates that choice of primary antibody and scoring system have a substantial impact on the determination of EGFR immunoreactivity.

Immunohistochemical detection of E-cadherin in differentiated thyroid carcinomas correlates with clinical outcome.

A retrospective immunohistochemical analysis of the adhesion molecule E-cadherin (E-CD) was performed in 112 differentiated thyroid carcinomas and 38 synchronous and 20 relapse metastases primarily from operations performed at the Medical School Hanover between 1982 and 1992. E-CD-specific antibody 5H9 was applied to paraffin-embedded tissues. All patients were clinically followed for a maximal period of 12 years. Lack of E-CD expression i.e., <5% of tumor cells positive) occurred in 18 of 112 (16.1%) cases, whereas the majority showed either low (24.1%), medium (35.7%), or high (24.1%) positivity. No difference was found between papillary (n = 88) and follicular (n = 24) carcinomas. Univariate statistical analysis for survival (Kaplan-Meier) showed that lack of E-CD expression (P < 0.024) is an adverse prognostic factor for differentiated thyroid carcinomas. The highest significance was seen among patients without lymph node involvement at first presentation (pN(0); P = 0.0068) and among females (P = 0.0033). Multivariate analysis (Cox model) indicated that E-CD staining is an independent prognostic factor (corrected risk factor, 3.7; P < 0.03) in addition to distant metastasis (pM1) and tumor size. A comparison of E-CD stainings between primary tumors and their metastatic lesions showed similar results in both synchronous and relapse metastases after therapy. In conclusion, E-CD immunostaining is an independent prognostic indicator for differentiated thyroid carcinomas. It may help to uncover the small group of patients with differentiated thyroid carcinomas carrying a high risk of suffering an unfavorable clinical outcome.

Resectional surgery of hilar cholangiocarcinoma: a multivariate analysis of prognostic factors.

PURPOSE: To define the prognostic factors after surgical resection of bile duct carcinomas at the hepatic bifurcation. PATIENTS AND METHODS: The retrospective single-center experience details 151 patients after surgical resection of central bile duct carcinoma performed between 1971 and 1995. Tumor removal was accomplished by resection of the bile duct bifurcation either alone (group I, n = 33), in combination with hepatic resection (group II, n = 77), or combined with hepatic and vascular resection (group III, n = 41). Survival analysis was performed by the Kaplan-Meier method and the relationship between each of the clinicopathologic variables and survival was assessed by the log-rank test. Multivariate results were confirmed using Cox regression. RESULTS: The overall hospital mortality rate was 9.9% and depended on the extent of resection (group 1, 6.1%; group II, 7.8%; group III, 17.1%). After exclusion of hospital deaths, the overall patient survival rate was 28.4% at 5 and 15.5% at 10 years, with a median survival duration of 2.05 +/- 0.23 years. Univariate survival analysis identified tumor size, lymph node metastases, residual tumor stage, and tumor grading as factors with a statistically significant prognostic impact. Survival prognosis was not influenced by the site of the tumor according to the classification of Bismuth and Corlette, extent of resection, International Union Against Cancer (UICC) stage, perineural and vascular invasion, age, or sex. In a multivariate Cox analysis, only lymph node metastases and residual tumor stage proved to be of independent prognostic significance. CONCLUSION: Resection of central bile duct carcinoma is feasible in many patients and a favorable outcome after resection is mainly determined by curative resection and the absence of lymph node metastases.

Pathobiology of NPM-ALK and variant fusion genes in anaplastic large cell lymphoma and other lymphomas.

Despite its clinical and histological heterogeneity, anaplastic large cell lymphoma (ALCL) is now a well-recognized clinicopathological entity accounting for 2% of all adult non-Hodgkin's lymphomas (NHL) and about 13% of pediatric NHL. Immunophenotypically, ALCL are of T cell (predominantly) or Null cell type; by definition, cases expressing B cell antigens are officially not included in this entity. The translocation (2;5)(p23;q35) is a recurring abnormality in ALCL; 46% of the ALCL patients bear this signature translocation. This translocation creates a fusion gene composed of nucleophosmin (NPM) and a novel receptor tyrosine kinase gene, named anaplastic lymphoma kinase (ALK). The NPM-ALK chimeric gene encodes a constitutively activated tyrosine kinase that has been shown to be a potent oncogene. The exact pathogenetic mechanisms leading to lymphomagenesis remain elusive; however, the synopsis of evidence obtained to date provides an outline of likely scenarios. Several t(2;5) variants have been described; in some instances, the breakpoints have been cloned and the genes forming a new fusion gene with ALK have been identified: ATIC-ALK, TFG-ALK and TPM3-ALK. Cloning the translocation breakpoint and identifying the ALK and NPM genes provided tools for screening material from patients with ALCL using various approaches at the chromosome, DNA, RNA, or protein level: positive signals in the reverse transcriptase-polymerase chain reaction (RT-PCR) and the immunostaining with anti-ALK monoclonal antibodies (McAb) serve as the most convenient tests for detection of the t(2;5) NPM-ALK since the fusion gene and ALK protein expression do not occur in normal or reactive lymphoid tissue. The wide range of NPM-ALK positivity reported in different series appears to be dependent on the inclusion and selection criteria of the ALCL cases studied. Overall, however, 43% of ALCL cases were NPM-ALK+ (83% of pediatric ALCL vs 31% of adult ALCL). Occasional non-ALCL B cell lymphomas (4%) with diffuse large cell and immunoblastic histology and Hodgkin's disease cases (3%) were NPM-ALK-, but these data are questionable. The aggregate results indicate that, in contrast to primary nodal (systemic) ALCL, the t(2;5) may be present in only 10-20% of primary cutaneous ALCL and rarely, if at all, in lymphomatoid papulosis, a potential precursor lesion; however, these 10-20% positive cases were not confirmed by anti-ALK McAb immunostaining and may represent an overestimate. Positivity for NPM-ALK is associated to various degrees with the following parameters: 44% and 45% of ALCL cases with T cell and Null cell immunophenotype, respectively, are positive, whereas only 8% of cases with a B cell immunoprofile are positive; the mean age of positive patients is significantly younger than that of negative patients; positive cases carry a better overall prognosis (but not in all studies). Recently, the homogenous category of ALK lymphoma ('ALKoma') has emerged as a distinct pathological entity within the heterogenous group of ALCL. The fact that patients with ALK lymphomas experience significantly better overall survival than ALK- ALCL demonstrates further that analysis of ALK expression has important prognostic implications. The term ALK lymphoma signifies a switch in the use of the diagnostic criteria: cases are selected on the basis of a genetic abnormality (the ALK rearrangement), instead of the review of morphological or immunophenotypical features which are clearly more prone to disagreement and controversy. Since its initial description in 1985 ALCL has become one of the best characterized lymphoma entities.

Identification of a Wnt/beta-catenin signaling pathway in human thyroid cells.

beta-Catenin is a structural component of the adherens junctions. Outside the adherens junctions a complex consisting of glycogen synthase kinase 3beta, the tumor suppressor adenomatous polyposis coli, and axin constantly targets beta-Catenin for degradation to keep levels of free beta-Catenin low. Free beta-Catenin is able to bind to transcription factors of the T cell factor/lymphoid-enhancing factor family and to stimulate transcription of target genes. This signaling function of beta-Catenin is activated by extracellular Wnt factors that bind to Frizzled receptors and induce inhibition of beta-Catenin degradation. By RT-PCR and subcloning, we observed the expression of five Wnt factors, three members of the Frizzled receptor family, and all known Disheveled isoforms in thyroid cells. Immunoprecipitation studies demonstrated the formation of the complex targeting beta-Catenin for degradation. Introduction of a degradation resistant beta-Catenin into the thyroid carcinoma cell line WRO induced appearance of monomeric beta-Catenin as shown by size fractionation and nuclear beta-Catenin immunostaining. Reporter gene assays demonstrated a stimulation of T cell factor/lymphoid-enhancing factor-mediated transcription in these cells. In ARO cells, a thyroid carcinoma cell line carrying a mutated adenomatous polyposis coli gene, monomeric beta-Catenin and nuclear immunostaining were observed. In summary, our data indicate that elements of the Wnt signaling pathway are expressed in thyroid cells and that this pathway is functionally active.

Clinical significance of E-cadherin as a prognostic marker in thyroid carcinomas.

The aim of this study was to evaluate the expression of E-cadherin as a potential marker for the prognosis of thyroid carcinomas. In normal thyroid (n = 8), the expression of E-cadherin messenger ribonucleic acid levels was uniformly high and seemed to be restricted to thyrocytes. Steady-state messenger ribonucleic acid levels and immunostaining were both completely lost in undifferentiated thyroid carcinomas (n = 7) and were variably reduced in differentiated thyroid carcinomas (n = 44). In a follow-up study during a mean of 4.5 +/- 1.4 yr, E-cadherin messenger ribonucleic acid and immunohistochemical expression were compared with the initial clinicopathological parameters and with locoregional recurrence and the development of nodal or distant metastases in differentiated thyroid carcinomas. Immunohistochemical expression of E-cadherin was greatly reduced with the progression to primary tumor stage 4 (pT4) tumors. In parallel, patients with pT4 tumors had a higher rate of locoregional tumor recurrence and distant metastasis than did the group of patients with pT1-3 tumors. In 5 of 29 patients with pT4 tumors, positive E-cadherin staining of more than 30% of the cells was detected. None of these patients showed signs of a regional recurrence or distant metastases during an observation period of 4.3 +/- 1.1 yr. In 13 patients with E-cadherin-positive tumors, none developed new distant metastases which was in contrast to 7 of the group of 31 patients with less than 30% E-cadherin-positive cells. Thus, E-cadherin expression seems to be associated with the dedifferentiation, progression, and metastatic spread of thyroid carcinomas and may be a useful marker for the prognosis of these tumors.

Infiltration of the central nervous system by a granulosa cell tumor immunostained by antibodies against inhibin.

Involvement of the CNS by a granulosa cell tumor of the ovary is rare. We describe a patient with leptomeningeal and cerebral cortex infiltration by this tumor. The diagnosis was confirmed by CSF cytology and immunocytochemistry using monoclonal antibodies against human inhibin.

Diagnostic impact of fluorescence in situ hybridization in the differentiation of hepatocellular adenoma and well-differentiated hepatocellular carcinoma.

Histopathological differentiation between hepatocellular adenoma and well differentiated hepatocellular carcinoma (HCC) may be a difficult task in small biopsies and occasionally in resected tumor specimens. Whether the analysis of chromosome aberrations can contribute to a more precise discrimination has not been analyzed systematically up to now. Therefore, fluorescence in situ hybridization was applied to 28 cases of adenoma and well differentiated carcinoma, using centromeric probes for chromosomes 1, 6, 7, 8, and X. None of 14 adenomas revealed an aberrant count in the analyses performed. By contrast, 13/14 carcinomas demonstrated aberrations for 2-5 chromosomes/case. Chromosome 1 was aberrant in 8/12 cases informative for this probe (67%), chromosomes 6 and 7 were aberrant in 9/14 cases (64%), chromosome 8 was aberrant in 11/14 cases (79%), and chromosome X in 7/14 cases (50%). Taking results for chromosomes 1 and 8 together, 13/14 HCC revealed aberrations for at least one of these chromosomes. Probes for 6, 7, and X revealed no additional aberrant cases.Thus, FISH for chromosomes 1 and 8, extended by probes for chromosomes 6, 7 and X, represents a promising approach toward a more accurate differentiation between hepatocellular adenoma and carcinoma.

Quality assurance in immunohistochemistry: results of an interlaboratory trial involving 172 pathologists.

The practicability of quality assurance in immunohistochemistry and its integration into the diagnostic process were both tested in this Germany-wide interlaboratory trial. One hundred seventy-two pathologists received one hematoxylin and eosin and five unstained slides from five cases; all cases were selected by a panel because immunohistochemistry was required for their final diagnosis. Participants rendered a morphologic diagnosis and then substantiated it immunohistochemically. Stained slides and evaluation sheets were reviewed by the panel, and the diagnostic process was analyzed in individual steps: morphologic diagnosis, selection of antibodies, staining quality, interpretation of stained slides, conclusions, and final diagnosis. Diagnosis-independent immunohistochemical performance was tested using a multisample tissue block (30 samples) that was stained and evaluated for six common antigens. For individual cases, corresponding to their difficulty, 21-89% of the final diagnoses (altogether 57% from 828 diagnoses) were correct. In a statistical analysis, the tentative diagnosis, the interpretation of stains and conclusions drawn from immunohistochemistry, were independent factors in reaching the diagnosis. Sensitivity to detect estrogen receptors on the multisample tissue block was only 48%. However, 24% of the stains were interpreted as falsely negative. The low staining sensitivity was not correlated to the number of correct diagnoses. The major problem of applying immunohistochemistry in surgical pathology appears to be its integration into the diagnostic process and not the staining quality. Both future quality control projects and training will have to regard these integrative requirements. Multisample tissue blocks provide a promising tool to standardize quantitative immunohistochemical parameters, such as receptor or proliferation scores.

Tyramine amplification technique in routine immunohistochemistry.

Signal amplification in immunohistochemistry via binding of biotinylated tyramine to proteins near the site of peroxidase-labeled antibodies is a promising new technique, but studies investigating a wide range of markers are lacking. The tyramine amplification technique (TAT) was investigated on 85 antibodies using a simple and fast protocol, and TAT results were compared to those obtained with conventional immunohistochemistry. Using TAT, most of the markers could be 5- to 50-fold further diluted and still showed identical staining results compared with standard stainings (maximal 500-fold). However, the variable reactivity of the different markers with TAT underlines the need for individual testing of every antibody to determine the optimal dilution. Some antibodies against cell adhesion molecules could be demonstrated for the first time in archival, formalin-fixed tissue sections. TAT, if carefully evaluated, offers a revolutionary improvement for modern immunostaining, either to increase sensitivity or primary antibody dilutions (cost reduction). From a methodological point of view, immunohistochemistry has not reached its limits by far and TAT is an important progressive step in this developmental process.

Aberrations of chromosomes 5 and 8 as recurrent cytogenetic events in anaplastic carcinoma of the thyroid as detected by fluorescence in situ hybridisation and comparative genomic hybridisation.

Comparative genomic hybridisation (CGH) is a technique which identifies gains and losses of DNA sequence copy number in tumours. We used CGH to search for genetic changes in one of the most aggressive malignancies--anaplastic thyroid carcinoma (ATC). For this purpose, we analysed tumour specimens of nine ATCs and DNA of two ATC cell lines. CGH detected aberrations in 10 of 11 samples, with a mean number of gains or losses per carcinoma of 4.8 (range 0-13). Total or partial changes of chromosome 8 (n=6), including gains or losses of 8p (n=6) or 8q (n=5) were those detected most frequently. Chromosome 5p was amplified in five cases. Gains in two of three samples were found for 3q, 7p, 11q and 20q. Gains in a fewer number were seen for 1p (1 case), 1q (1), 7q (2), 9q (2), 11p (2), 12q (1), 14 (1), 15 (1), 17q (2), 18p (2), 18q (1), 20p (1), 21 (2), Xp (2) and Xq (2). Losses were less frequent than gains and observed for 1p (2 cases), 1q (1), 2p (1), 2q (2), 3p (2), 3q (1), 4q (2), 6q (1), 9p (2), 9q (1), 18p (1), 18q (1) and Y (2). Examples of analysis of tumour sections and cell lines performed by fluorescence in situ hybridisation (FISH) confirmed the gains and losses found by CGH and detected additional signals for 8q21 in tumour cells in a sample with no gains or losses normally in CGH. The results suggest that aberrations of 5p, 8p and 8q, which are rarely found in differentiated thyroid carcinoma, may play an important role in the development of ATC. Therefore, these chromosomes could harbour gene loci potentially involved in the aggressiveness of neoplastic tumours, as shown in tumours such as in this study for ATC.

Megakaryocytes carry the fused bcr-abl gene in chronic myeloid leukaemia: a fluorescence in situ hybridization analysis from bone marrow biopsies.

Histological examination of bone marrow biopsies shows that about one-third of chronic myeloid leukaemia (CML) patients exhibit an increase of megakaryocytes. The megakaryocytic predominance may be so striking that differentiation from other chronic myeloproliferative disorders (CMPD) may be difficult in some CML patients. Megakaryocytes in CML are clonal as demonstrated by loss of glucose-6-phosphate dehydrogenase isoenzymes. The Ph translocation, fusing the abl and bcr genes on chromosomes 9 and 22, however, obviously occurs as a second step in tumour development. So far, the Ph translocation has not been assigned explicitly to megakaryocytes. The question is whether the megakaryocytic cell lineage could harbour the bcr/abl fusion in those CML cases with striking proliferation of megakaryocytes but lack this genetic defect in cases with normal or decreased megakaryocyte counts. We therefore performed triple-colour fluorescence in situ hybridization (FISH) for portions of the bcr and abl genes flanking the breakpoint in CML in paraffin sections of CML cases with normal and with increased numbers of megakaryocytes. This method allows identification of the bcr/abl fusion in single, morphologically intact cells, whereas conventional cytogenetics requires lysis and thus destruction of the cell. Among the 21 CML patients examined by FISH, 10 were informative for bcr and abl genes and displayed distinct hybridization signals within nuclei of bone marrow cells. Besides the granulopoietic cells, megakaryocytes of all those patients (4 without and 6 with varying grades of megakaryocytic increase) displayed bcr/abl fusion signals indicative of a Ph translocation. The lack of hybridization signals in the remaining 11 cases indicates that this technique is not of value diagnostically and should be reserved for scientific questions. Positive controls consisted of conventional chromosome preparations from bone marrow aspirates demonstrating the Ph chromosome in all patients examined, and negative controls of paraffin sections of bone marrow biopsies from non-CML patients. These showed no fusion signals in bone marrow cells, including megakaryocytes, using FISH. Our results demonstrate clearly that not only the transforming event but also the Ph translocation leading to the bcr/abl fusion happens prior to the differentiation of the pluripotent stem cell into different myeloid lineages. The megakaryocytic proliferation evident in some CML cases is probably a consequence of the disease progress.

Expression of proliferation associated antigens and detection of numerical chromosome aberrations in primary human liver tumours: relevance to tumour characteristics and prognosis.

AIMS: To assess cell proliferation and the presence of numerical chromosome aberrations involving chromosomes 1 and 8 in benign and malignant liver tumours. METHODS: Cell proliferation was studied immunohistochemically in paraffin wax embedded material from 62 primary liver tumours (20 hepatocellular carcinomas, 16 cholangiocellular carcinomas, 15 liver cell adenomas, 11 focal nodular hyperplasias), and the results were compared with histological characteristics and clinical data. Copy numbers of chromosomes 1 and 8 were assessed by interphase fluorescence in situ hybridisation (FISH) with satellite probes in fresh tumour material. RESULTS: The expression of proliferation associated antigen Ki67, using the monoclonal antibody MIB-1, and proliferating cell nuclear antigen (PCNA), using the antibody PC10, was found to be significantly higher in malignant versus benign liver tumours. Neither Ki67 nor PCNA expression were independent prognostic parameters. However, there was a tendency for a worse outcome (survival < 12 months) for patients with a high MIB-1 labelling index (> 20%) compared with patients having the same tumour stage and a low MIB-1 index. Aneusomy for chromosomes 1 and 8 was demonstrated by FISH in malignant tumours (six of seven hepatocellular carcinomas, four of five cholangiocellular carcinomas) but not in benign tumours (none of nine) or non-neoplastic liver (none of nine). CONCLUSION: Both the determination of the proliferating cell fraction and FISH analysis are useful for distinguishing hepatocellular carcinoma from liver cell adenoma or focal nodular hyperplasia; high fractions of proliferating cells are predictive of an early relapse.

The bcl-2/JH gene rearrangement is undetectable in Hodgkin's lymphomas: results from the German Hodgkin trial.

Ninety-one Hodgkin's lymphomas (HD), 52 non-Hodgkin lymphomas (NHL) and 33 specimens of non-neoplastic lymphatic tissues were investigated by polymerase chain reaction (PCR) for the presence of the bcl-2/JH gene rearrangement. The majority of the HD cases were drawn from the files of the German Hodgkin trial where diagnoses are established by a panel of four independent histopathologists. Using the very sensitive PCR method which detected 1 positive among 10000 negative cells, the bcl-2/JH gene rearrangement was found in 7/52 NHL and 3/16 tonsils with follicular hyperplasia, but in none of the 91 HD. The bcl-2 protein, however, was expressed by malignant cells of B and T cell lymphomas and by the giant tumour cells in 2/13 HD lymphocyte predominant, 11/28 HD nodular sclerosing I, 14/17 HD nodular sclerosing II, 10/27 HD mixed cellularity and 3/3 HD lymphocyte depleted. The bcl-2/JH rearrangement is thus independent of protein over-expression, the latter being found in all types of lymphomas. Our results do not confirm the findings of others who have detected the bcl-2/JH rearrangement in HD. These discrepancies may be explained by differences in choice of material, the gene rearrangement actually occurring in bystander cells but not in Reed-Sternberg or Hodgkin cells, or by contamination.