Konradin Metze

Usefulness of HBME-1, cytokeratin 19 and galectin-3 immunostaining in the diagnosis of thyroid malignancy

Aims : To investigate the usefulness of immunohistochemical expression and immunolocalization of a panel of thyroid malignancy markers including HBME‐1, cytokeratin (CK) 19 and galectin‐3.

Chronic nitric oxide inhibition as a model of hypertensive heart muscle disease

We have compared the myocardial alterations in rats made hypertensive by the chronic inhibition of nitric oxide biosynthesis with those having renal hypertension (two kidney-one clip model). Male Wistar rats were chronically administered the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) for 2, 4 and 8 weeks. Both groups initially developed a similar increase in blood pressure but only the 2K-1C rats developed myocardial hypertrophy after 2–4 weeks. L-NAME-treated animals developed a similar degree of hypertrophy following 8 weeks of treatment. As observed by light microscopy, the myocardial alterations in the latter animals consisted of extensive areas of fibrosis and myocardial necrosis, especially in regions of the subendocardium. The histological alterations induced by L-NAME were not caused by the accompanying hypertension, since the 2K-1C animals had a similar increase in arterial blood pressure without any significant alterations in the heart morphology. 2K-1C rats treated chronically with L-NAME behaved in a manner similar to the L-NAME-treated animals with regard to both the blood pressure increases and cardiac morphological alterations. Animals which received the inactive enantiomer D-NAME did not develop hypertension nor did they have any morphological abnormalities. Both the coronary flow and the contractile capacity of hearts isolated from rats treated with L-NAME for 8 weeks were impaired compared to control animals. These results indicate that the chronic inhibition of NO biosynthesis causes cardiac ischemia associated with a mechanical dysfunction that is unrelated to cardiac hypertrophy which is similar to those seen in some patients suffering from chronic arterial hypertension.

Fractal dimension of chromatin is an independent prognostic factor for survival in melanoma

BackgroundPrognostic factors in malignant melanoma are currently based on clinical data and morphologic examination. Other prognostic features, however, which are not yet used in daily practice, might add important information and thus improve prognosis, treatment, and survival. Therefore a search for new markers is desirable. Previous studies have demonstrated that fractal characteristics of nuclear chromatin are of prognostic importance in neoplasias. We have therefore investigated whether the fractal dimension of nuclear chromatin measured in routine histological preparations of malignant melanomas could be a prognostic factor for survival.MethodsWe examined 71 primary superficial spreading cutaneous melanoma specimens (thickness ≥ 1 mm) from patients with a minimum follow up of 5 years. Nuclear area, form factor and fractal dimension of chromatin texture were obtained from digitalized images of hematoxylin-eosin stained tissue micro array sections. Clarks level, tumor thickness and mitotic rate were also determined.ResultsThe median follow-up was 104 months. Tumor thickness, Clarks level, mitotic rate, nuclear area and fractal dimension were significant risk factors in univariate Cox regressions. In the multivariate Cox regression, stratified for the presence or absence of metastases at diagnosis, only the Clark level and fractal dimension of the nuclear chromatin were included as independent prognostic factors in the final regression model.ConclusionIn general, a more aggressive behaviour is usually found in genetically unstable neoplasias with a higher number of genetic or epigenetic changes, which on the other hand, provoke a more complex chromatin rearrangement. The increased nuclear fractal dimension found in the more aggressive melanomas is the mathematical equivalent of a higher complexity of the chromatin architecture. So, there is strong evidence that the fractal dimension of the nuclear chromatin texture is a new and promising variable in prognostic models of malignant melanomas.

Enalapril does not prevent the myocardial ischemia caused by the chronic inhibition of nitric oxide synthesis

In rats, chronic administration of the nitric oxide (NO) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) causes arterial hypertension, cardiac hypertrophy and myocardial ischemic alterations such as necrosis and fibrosis. In this study, we evaluated the effect of 8 weeks of treatment with enalapril maleate on cardiac weight and on the development of the histological alterations induced by L-NAME. Enalapril significantly inhibited the development of both arterial hypertension (117.2 +/- 5.8, 161.8 +/- 8.8 and 122.0 +/- 10.6 mm Hg, for control, L-NAME- and L-NAME + enalapril-treated animals, respectively) and left ventricular hypertrophy (1.36 +/- 0.13, 1.60 +/- 0.04 and 1.48 +/- 0.05 mg/g, for control, L-NAME- and L-NAME + enalapril-treated animals, respectively), but had no effect on the myocardial lesions. These findings demonstrate that although the renin-angiotensin system plays a major role in the development of arterial hypertension and cardiac hypertrophy, it does not modulate the ischemia-induced myocardial alterations observed in this model.

Fractal Characteristics of May-Grünwald-Giemsa Stained Chromatin Are Independent Prognostic Factors for Survival in Multiple Myeloma

Background The use of computerized image analysis for the study of nuclear texture features has provided important prognostic information for several neoplasias. Recently fractal characteristics of the chromatin structure in routinely stained smears have shown to be independent prognostic factors in acute leukemia. In the present study we investigated the influence of the fractal dimension (FD) of chromatin on survival of patients with multiple myeloma. Methodology We analyzed 67 newly diagnosed patients from our Institution treated in the Brazilian Multiple Myeloma Study Group. Diagnostic work-up consisted of peripheral blood counts, bone marrow cytology, bone radiograms, serum biochemistry and cytogenetics. The International Staging System (ISS) was used. In every patient, at least 40 digital nuclear images from diagnostic May-Grünwald-Giemsa stained bone marrow smears were acquired and transformed into pseudo-3D images. FD was determined by the Minkowski-Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R2 values in the log-log plots. The influence of diagnostic features on overall survival was analyzed in Cox regressions. Patients that underwent autologous bone marrow transplantation were censored at the day of transplantation. Principal Findings Median age was 56 years. According to ISS, 14% of the patients were stage I, 39% were stage II and 47% were stage III. Additional features of a bad prognosis were observed in 46% of the cases. When stratifying for ISS, both FD and its goodness-of-fit were significant prognostic factors in univariate analyses. Patients with higher FD values or lower goodness-of-fit showed a worse outcome. In the multivariate Cox-regression, FD, R2, and ISS stage entered the final model, which showed to be stable in a bootstrap resampling study. Conclusions Fractal characteristics of the chromatin texture in routine cytological preparations revealed relevant prognostic information in patients with multiple myeloma.

The Fractal Dimension of Nuclear Chromatin as a Prognostic Factor in Acute Precursor B Lymphoblastic Leukemia

The fractal nature of the DNA arrangement has been postulated to be a common feature of all cell nuclei. We investigated the prognostic importance of the fractal dimension (FD) of chromatin in blasts of patients with acute precursor B lymphoblastic leukemia (B-ALL). In 28 patients, gray scale transformed pseudo-3D images of 100 nuclei (May–Grünwald–Giemsa stained bone marrow smears) were analyzed. FD was determined by the Minkowski–Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R2 values in the log-log plots. Whereas FD presented no prognostic relevance, patients with higher R2 values showed a prolonged survival. White blood cell count (WBC), age and mean fluorescence intensity of CD45 (MFICD45) were all unfavorable prognostic factors in univariate analyses. In a multivariate Cox-regression, R2, WBC, and MFICD45, entered the final model, which showed to be stable in a bootstrap resampling study. Blasts with lower R2 values, equivalent to accentuated “coarseness” of the chromatin pattern, which may reflect profound changes of the DNA methylation, indicated a poor prognosis. In conclusion the goodness-of-fit of the Minkowski–Bouligand dimension of chromatin can be regarded as a new and biologically relevant prognostic factor for patients with B-ALL.

Lymph vascular invasion in invasive mammary carcinomas identified by the endothelial lymphatic marker D2-40 is associated with other indicators of poor prognosis

BackgroundImmunohistochemical studies of lymphatic vessels have been limited by a lack of specific markers. Recently, the novel D2-40 antibody, which selectively marks endothelium of lymphatic vessels, was released. The aim of our study is to compare lymphatic and blood vessel invasion detected by hematoxylin and eosin (H&E) versus that detected by immunohistochemistry, relating them with morphologic and molecular prognostic factors.MethodsWe selected 123 cases of invasive mammary carcinomas stratified into three subgroups according to axillary lymph node status: macrometastases, micrometastases, and lymph node negative. Lymphatic (LVI) and blood (BVI) vessel invasion were evaluated by H&E and immunohistochemistry using the D2-40 and CD31 antibodies, and related to histologic tumor type and grade, estrogen and progesterone receptors, E-cadherin, Ki67, p53, and Her2/neu expression.ResultsLVI was detected in H&E-stained sections in 17/123 cases (13.8%), and in D2-40 sections in 35/123 cases (28.5%) (Kappa = 0.433). BVI was detected in H&E-stained sections in 5/123 cases (4.1%), and in CD31 stained sections in 19/123 cases (15.4%) (Kappa = 0.198). LVI is positively related to higher histologic grade (p = 0.013), higher Ki67 expression (p = 0.00013), and to the presence of macrometastases (p = 0.002), and inversely related to estrogen (p = 0.0016) and progesterone (p = 0.00017) receptors expression.ConclusionD2-40 is a reliable marker of lymphatic vessels and is a useful tool for lymphatic emboli identification in immunostained sections of breast carcinomas with higher identification rates than H&E. Lymphatic vessel invasion was related to other features (high combined histologic grade, high Ki67 score, negative hormone receptors expression) associated with worse prognosis, probable reflecting a potential for lymphatic metastatic spread and aggressive behavior.

Fractal dimension of chromatin: potential molecular diagnostic applications for cancer prognosis

Fractal characteristics of chromatin, revealed by light or electron microscopy, have been reported during the last 20 years. Fractal features can easily be estimated in digitalized microscopic images and are helpful for diagnosis and prognosis of neoplasias. During carcinogenesis and tumor progression, an increase of the fractal dimension (FD) of stained nuclei has been shown in intraepithelial lesions of the uterine cervix and the anus, oral squamous cell carcinomas or adenocarcinomas of the pancreas. Furthermore, an increased FD of chromatin is an unfavorable prognostic factor in squamous cell carcinomas of the oral cavity and the larynx, melanomas and multiple myelomas. High goodness-of-fit of the regression line of the FD is a favorable prognostic factor in acute leukemias and multiple myelomas. The nucleus has fractal and power-law organization in several different levels, which might in part be interrelated. Some possible relations between modifications of the chromatin organization during carcinogenesis and tumor progression and an increase of the FD of stained chromatin are suggested. Furthermore, increased complexity of the chromatin structure, loss of heterochromatin and a less-perfect self-organization of the nucleus in aggressive neoplasias are discussed.

Letter to the Editor: The Importance of the Mitotic Index as a Prognostic Factor for Survival of Canine Cutaneous Mast Cell Tumors: A Validation Study

Editor: The importance of the mitotic index (MI) as a prognostic factor in veterinary oncology has been emphasized recently. In their interesting exploratory study on canine cutaneous mast cell tumors, Romansik et al. showed a significant association between MI and overall survival, which could be important for clinical decision making, especially in the case of Patnaik grade II tumors. The median survival time for dogs with a tumor MI # 5 was significantly longer than for those with a MI . 5, regardless of the grade. Similarly, dogs with Patnaik grade II tumors and a MI # 5 survived longer than those with a MI . 5. The cut-point applied in this study was based on the distribution of the MI, as well as on MI categories published for other tumor types. Because the choice of the cut-point is critical in prognostic factor studies and because the clinical relevance of new prognostic indicators should always be investigated in an independent group, we started a validation study to test the utility of the cut-points suggested by Romansik et al. Fifty-seven dogs with cutaneous mast cell tumors were included in this study. In every case, the diagnosis had been confirmed independently by 3 veterinary pathologists, and complete follow-up information had been obtained. The tumor grade was determined according to Patnaik by consensus of the 3 observers. The number of mitotic figures/10 high-power fields was counted as described by Romansik et al. and compared with survival data in a Cox proportional hazards model using MI as a stratified variable. The mean MI of grade 1 tumors (n 5 5) was 0.6 (range: 0–3). Grade II tumors (n 5 41) had a mean MI of 2.2 (range: 0–16); grade III tumors (n 5 11) had a mean MI of 7.4 (range: 0–21; Kruskal-Wallis test P 5 .0033). The Dunn posthoc test showed significant differences between grades I and II and between I and III but not between II and III. Furthermore, a Jonckheere-Terpstra test demonstrated that the MI values increased with grade (P 5 .001). When applying a cut-point at MI 5 5, our results were very similar (Figs. 1, 2) to those reported by Romansik et al. Dogs with an MI . 5 had a median survival time of 8 months, whereas dogs with MI # 5 did not reach median survival time (P 5 .003; log-rank test). Next, we looked for other cut-points, submitting the MI values to a cluster analysis according to the Ward algorithm after a logarithmic transformation. Furthermore, the R values, also called coefficients of determination, of each Cox regression were recorded. R varies between 0.0 and 1.0 and can be interpreted as the fraction of the total variance of the survival regression, which is explained by the MI score system. Finally, the stability of the regressions was tested by bootstrap resampling. This technique creates new data sets of equal size by random sampling of the original data with replacement. Then for each of these ‘‘new’’ sets a new Cox regression is calculated and the variables entering the ‘‘new’’ regressions are recorded. WinStat 3.1 and SPSS 8.0 software was used. The dendrogram derived from the cluster analysis strongly suggested the creation of 3 groups: 1) dogs with tumor MI 5 0, which did not reach median survival; 2) dogs with tumor MI between 1 and 7 and a median survival time of 18 months; and 3) dogs with tumor MI . 7 and a Fig. 1. Kaplan-Meier survival plot of all dogs (n 5 57). Stratification at a cut-point of MI 5 5. Log-rank test P 5 .003.

The impact of maternal HIV infection on cord blood lymphocyte subsets and cytokine profile in exposed non-infected newborns

BackgroundChildren born to HIV+ mothers are exposed intra-utero to several drugs and cytokines that can modify the developing immune system, and influence the newborns immune response to infections and vaccines. We analyzed the relation between the distribution of cord blood lymphocyte subsets and cytokine profile in term newborns of HIV+ mothers using HAART during pregnancy and compared them to normal newborns.MethodsIn a prospective, controlled study, 36 mother-child pairs from HIV+ mothers and 15 HIV-uninfected mothers were studied. Hematological features and cytokine profiles of mothers at 35 weeks of pregnancy were examined. Maternal and cord lymphocyte subsets as well as B-cell maturation in cord blood were analyzed by flow cytometry. The non-stimulated, as well as BCG- and PHA-stimulated production of IL2, IL4, IL7, IL10, IL12, IFN-γ and TNF-alpha in mononuclear cell cultures from mothers and infants were quantified using ELISA.ResultsAfter one year follow-up none of the exposed infants became seropositive for HIV. An increase in B lymphocytes, especially the CD19/CD5+ ones, was observed in cord blood of HIV-exposed newborns. Children of HIV+ hard drug using mothers had also an increase of immature B-cells. Cord blood mononuclear cells of HIV-exposed newborns produced less IL-4 and IL-7 and more IL-10 and IFN-γ in culture than those of uninfected mothers. Cytokine values in supernatants were similar in infants and their mothers except for IFN-γ and TNF-alpha that were higher in HIV+ mothers, especially in drug abusing ones. Cord blood CD19/CD5+ lymphocytes showed a positive correlation with cord IL-7 and IL-10. A higher maternal age and smoking was associated with a decrease of cord blood CD4+ cells.Conclusionsin uninfected infants born to HIV+ women, several immunological abnormalities were found, related to the residual maternal immune changes induced by the HIV infection and those associated with antiretroviral treatment. Maternal smoking was associated to changes in cord CD3/CD4 lymphocytes and maternal hard drug abuse was associated with more pronounced changes in the cord B cell line.