The Fractal Dimension Suggests Two Chromatin Configurations in Small Cell Neuroendocrine Lung Cancer and Is an Independent Unfavorable Prognostic Factor for Overall Survival.

Experimental studies have shown that in small cell neuroendocrine lung carcinomas (SCLC) global opening of the chromatin structure is associated with a higher transcription activity and increase of tumor aggressiveness and metastasis. The study of the fractal characteristics (FD) of nuclear chromatin has been widely used to describe the cell nuclear texture and its changes correspond to changes in nuclear metabolic and transcription activity. Hence, we investigated whether the nuclear fractal dimension could be a prognostic factor in SCLC. Hematoxylin-eosin stained brush cytology slides from 49 patients with SCLC were retrieved from our files. The chromatin (FD) was calculated in digitalized and interactively segmented nuclei using a differential box-counting method. The 3,575 nuclei studied showed a bimodal distribution (peaks at FD1 = 2.115 and FD2 = 2.180). The 75 percentile of the FD was an independent unfavorable prognostic factor for overall survival when tested together with ECOG (Eastern Cooperative Oncology Group) performance status, tumor extension, and therapy in a multivariate Cox regression. Our study corroborates the concept of two main chromatin configurations in small cell neuroendocrine carcinomas and that globally more open chromatin indicates a higher risk of metastasis and therefore a shorter survival of the patient.

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

BACKGROUND: Prognostic 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. METHODS: We examined 71 primary superficial spreading cutaneous melanoma specimens (thickness > or = 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. Clark's level, tumor thickness and mitotic rate were also determined. RESULTS: The median follow-up was 104 months. Tumor thickness, Clark's 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. CONCLUSION: In 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.

Perforin and granzyme B involvement in oral lesions of lichen planus and chronic GVHD.

BACKGROUND: Oral lesions of lichen planus and chronic graft-vs.-host disease (cGVHD) have similar clinical and histological features, but distinct etiology. Apoptosis induced by cytotoxic T lymphocyte has been proposed as a mechanism of keratinocytes death. Cytotoxicity can be mediated by granules containing granzyme B and perforin. Since common features can reflect similarities in immunological mechanisms, we studied the role of those molecules in both diseases. METHODS: We analyzed 29 cases of oral lichen planus and 27 of oral cGVHD. The sections were studied on H&E, perforin and granzyme B staining. RESULTS: The total means (epithelium plus connective tissue number) of the granzyme B- and perforin-positive cells were significantly higher in cGVHD than in oral lichen planus lesions (P<0.05). Also, it was found that the higher the number of perforin+ cells, the higher the number of granzyme-B+ cells in the epithelium and in the connective tissue for both groups (P < 0.05). In oral lichen planus, the number of single apoptotic bodies had a positive correlation with connective tissue granzyme immunostaining and a negative correlation with perforin (P<0.01). On the contrary, in oral cGVHD, the number of apoptotic body clusters presented a positive correlation with connective tissue perforin (P<0.01). CONCLUSIONS: Our findings indicate that apoptosis in oral lichen planus seems to be correlated with granzyme B release, while in oral cGVHD, perforin seems to be more important. Although these diseases present clinical and histological similarities, subtle differences seem to exist in their pathogenetic mechanisms.

The fractal dimension of chromatin - a potential molecular marker for carcinogenesis, tumor progression and prognosis.

INTRODUCTION: Fractality is omnipresent in medicine and life sciences. In particular, the fractal principle is found simultaneously at different organization levels of the cell nucleus. The aim of this review is to show whether fractal characteristics of chromatin could be related to tumor pathology and pathophysiology. Areas covered: This review provides an overview of the application of fractal measurements of chromatin or DNA for the characterization of physiological or pathological processes, in particular for the detection of preneoplastic changes, the characterization of tumor progression, the differential diagnosis between neoplasms and for prognosis. We used a network-based literature research strategy, i.e. after a systematic investigation by key-words, we looked for all citations (and the citations to these citations) of the selected papers in Scopus and Webofscience. Expert opinion: The fractal dimension (FD) increases during carcinogenesis, thus permitting the diagnosis of malignancy. In various malignant tumors, a higher FD or diminished goodness-of-fit of its regression line indicates a more aggressive behavior and worse prognosis. Applying new spectral techniques, the chromatin FD can be estimated at scales below the light microscopic resolution. The latter also permits the examination of live cells and studies on field carcinogenesis and chemoprophylaxis.

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.

Biomineralization of polyanionic collagen-elastin matrices during cavarial bone repair.

The polyanionic collagen-elastin matrices (PCEMs) are osteoconductive scaffolds that present high biocompatibility and efficacy in the regeneration of bone defects. In this study, the objective was to determine if these matrices are directly mineralized during the osteogenesis process and their influence in the organization of the new bone extracellular matrix. Samples of three PCEMs, differing in their charge density, were implanted into critical-sized calvarial bone defects created in rats and evaluated from 3 days up to 1 year after implantation. The implanted PCEMs were directly biomineralized by osteoblasts as shown by ultrastructural, histoenzymologic, and morphologic analysis. The removal of the implants occurred during the bone remodeling process. The organization of the new bone matrix was evaluated by image texture analysis determining the Shannon's entropy and the fractal dimension of digital images. The bone matrix complexity decreased as the osteogenesis progressed approaching the values obtained for the original bone structure. These results show that the PCEMs allow faster formation of new bone by direct biomineralization of its structure and skipping the biomaterial resorption phase.

Computerized texture analysis of atypical immature myeloid precursors in patients with myelodysplastic syndromes: an entity between blasts and promyelocytes.

BACKGROUND: Bone marrow (BM) blast count is an essential parameter for classification and prognosis of myelodysplastic syndromes (MDS). However, a high degree of cell atypias in bone marrow hemopoietic cells may be found in this group of clonal disorders, making it difficult to quantify precisely myeloblasts, and to distinguish them from promyelocytes and atypical immature myeloid precursors. Our aim was to investigate whether computerized image analysis of routine cytology would help to characterize these cells. METHODS: In May-Grünwald-Giemsa stained BM smears of 30 newly diagnosed MDS patients and 19 cases of normal BM, nuclei of blasts and promyelocytes were digitalized and interactively segmented. The morphological classification of the cells was done by consensus of two observers. Immature granulocytic precursors, which could not be clearly classified either as blasts or promyelocytes, were called "atypic myeloid precursors". Nuclear morphometry and texture features derived from the co-occurrence matrix and fractal dimension (FD) were calculated. RESULTS: In normal BM, when compared to myeloblasts, nuclei of promyelocytes showed significant increase in perimeter and local texture homogeneity and a decrease in form factor, chromatin gray levels, Haralick's entropy, inertia, energy, contrast, diagonal moment, cluster prominence, the fractal dimension according to Minkowski and its goodness-of-fit. Compared to normal myeloblast nuclei, the chromatin texture of MDS myeloblasts revealed higher local homogeneity and goodness-of-fit of the FD, but lower values of entropy, contrast, diagonal moment, and fractal dimension. The same differences were found between nuclei of normal promyelocytes and those of MDS. Nuclei of atypical myeloid precursors showed intermediate characteristics between those of blasts and promyelocytes according to the quantitative features (perimeter, form factor, gray level and its standard deviation), but were similar to promyelocytes according to the texture variables inertia, energy, contrast, diagonal moment, cluster prominence, and Minkowski's fractal dimension. CONCLUSION: BM atypical immature myeloid precursors are difficult to be correctly classified in routine cytology. Although their cytoplasm is more similar to that of myeloblasts, computerized texture analysis indicates a nuclear chromatin remodeling more close to the promyelocyte, thus indicating an asynchronous intermediate maturation stage between blast and promyelocyte.

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 R(2) 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, R(2), 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.

[Contribution of nuclear texture analysis for the differential diagnosis of follicular lesions of the thyroid: comparison to immunohistochemical markers]. / Contribuição da análise de textura do núcleo celular para o diagnóstico diferencial de lesões foliculares da tireoide: comparação com marcadores imunoistoquímicos.

OBJECTIVE AND METHODS: To investigate the utility of nuclear chromatin texture assessment in the differential diagnosis of follicular patterned lesions, by means of examining 76 samples previously submitted to the immunohistochemical protein analysis of HBME-1, CK-19 and galectina-3. RESULTS: HBME-1 confirmed to be the most sensitive marker of malignancy. A series of morphometric, densitometric and texture variables were useful in the discrimination of the different types of follicular lesions. Among these variables, r(2), a parameter related to the granularity of the nucleus presented the best accuracy, sensitivity, specificity and positive and negative predictive values, distinguishing benign from malignant lesions. CONCLUSION: The morphometric analysis of nuclear chromatin images may add accuracy to the differential diagnosis of follicular patterned lesions.

Image preprocessing improves Fourier-based texture analysis of nuclear chromatin.

OBJECTIVE: To investigate whether preprocessing of digitized images can improve the image analysis of chromatin of cytologic preparations using Fast Fourier Transformation (FFT). STUDY DESIGN: In a preprocessing step the nuclear borders of the segmented nuclei were smoothed, thus avoiding the Airy ring artifact. We tested this method, comparing the inertia values of digitalized cardiomyocyte nuclei of rats of different ages. Furthermore, we created in silicio nuclear images with chromatin alterations at or nearby the nuclear edge in order to investigate the robustness of our method. RESULTS: After preprocessing, the FFT-derived variable inertia discriminated significantly better the chromatin structure of the nuclei at different ages in every frequency range. The investigation on simulated nuclei revealed that within the frequency ranges from 1.8 microm to 0.72 microm smoothing of the borders does not interfere with the detection of chromatin changes at the nuclear border. CONCLUSION: Smoothing of borders in segmented images can improve the analysis of Fourier-derived variables of the chromatin texture.

Shannon's entropy and fractal dimension provide an objective account of bone tissue organization during calvarial bone regeneration.

The regeneration of compact bone involves the deposition of a poorly organized connective tissue template that remodels into compact lamellar bone. An objective description of this process is difficult because classical histomorphometry is unable to correctly characterize qualitative changes in tissue complexity. In this study, we demonstrated the use of two distinct methods of image texture analysis, the Shannon's entropy [standard error (SE)], and the fractal dimension (FD) to characterize the formation and remodeling of newly formed compact bone within two different polyanionic collagen-elastin matrices. The matrices were implanted in defects created into parietal bones of rats. The SE and FD were calculated for histological images of the experimental groups collected 3, 7, 15, 30, 60, and 365 days postsurgery and for the original bone only at day 365. Results showed that the SE and the FD initially increased and then diminished for all groups from day 3 to day 365 approaching the values of the original bone. These results are consistent with poor tissue organization during early osteogenesis that remodels into an organized lamellar structure, showing that these methods can be valuable tools to describe bone tissue remodeling during the regeneration process of compact bones.

Phenotypic subtypes of acute lymphoblastic leukemia associated with different nuclear chromatin texture.

OBJECTIVE: To determine if phenotypic subtypes of acute lymphoblastic leukemia (ALL) are associated with different nuclear textures. STUDY DESIGN: In 49 newly diagnosed patients, diagnostic work-up was made by routinely Giemsa-stained smears and immunophenotyping. B-precursor ALL was further subdivided by European Group for the Immunological Classification of Leukemias criteria. T-ALL was analyzed as a whole group. One hundred nuclear images were acquired; standard morphometric variables and texture features derived from the co-occurrence matrix were calculated. RESULTS: In T-ALL, nuclei presented higher mean and minimal gray levels and higher local homogeneity and angular second moment but lower entropy values, contrast, diagonal moment and cluster prominence than did nuclei in B-derived ALL. In T-ALL, peripheral blood (PB) leukocyte count showed significant positive correlation with minimal gray level and inverse correlation with nuclear area. In B-ALL, peripheral leukocyte count showed positive correlation with mean fluorescence intensity of CD45. In T-ALL but not in B-ALL, inverse correlation existed among age and PB leukocyte count and mean gray levels, and direct correlation existed with nuclear area and mean optical density. CONCLUSION: ALL of B- or T-origin presented significant differences in nuclear texture features, probably reflecting different molecular events associated with cell differentiation, gene methylation pattern, apoptosis, and lineage-specific functional events.

Texture analysis of the epidermis based on fast Fourier transformation in Sjögren-Larsson syndrome.

OBJECTIVE: To investigate whether image analysis of routine hematoxylin-eosin (H-E) skin sections using fast Fourier transformation (FFT) could detect structural alterations in patients with Sjögren-Larsson syndrome (SLS) diagnosed by molecular biology. STUDY DESIGN: Skin punch biopsies of 9 patients with SLS and 17 healthy volunteers were obtained. Digital images of routine histologic sections were taken, and their gray scale luminance was analyzed by FFT. The inertia values were determined for different ranges of the spatial frequencies in the vertical and horizontal direction. To get an estimation of anisotropy, we calculated the resultant vector of the designated frequency ranges. RESULTS: In the prickle cell layer, SLS patients showed more intense amplitudes in spatial structures with periods between 1.2 and 3.6 microm in the vertical direction, which correlated in part with accentuated nuclei and nucleoli and perinucleolar halos in the H-E sections. In a linear discriminant analysis, the variables derived from the FFT images correctly discriminated 84.6% of the patients. Texture features derived from the gray level cooccurrence matrix were not able to separate the groups. CONCLUSION: Exploratory texture analysis by FFT was able to detect discrete alterations in the prickle cell layer in routine light microscopy slides of SLS patients. The structural changes identified by FFT may be related to abnormal cellular components associated with aberrant lipid metabolism.