DNA measurements in thin sections of lymphomas

The DNA content of the nuclei of lymphomas and a reactive lymph node was studied by light absorption measurements in Feulgen-stained thin (2 microns) sections of lymph node biopsies, using the TAS image analysis system. For each nucleus, the integrated light absorbance at 548 nm wavelength was multiplied by the square root of the nuclear area to obtain a parameter for DNA independent from the nuclear size. In a hyperplastic reactive lymph node with follicular hyperplasia, the distribution of DNA X square root area was different in centrocytes and centroblasts, being compatible with a diploid centrocyte fraction and a mainly tetraploid centroblast fraction, in which hypertetraploid (octoploid?) nuclei were present. Similar measurements in three follicular centroblastic-centrocytic lymphomas gave similar results, with the centroblasts mainly tetraploid. The DNA distribution was studied in sections of lymphomas of high and low grades of malignancy, classified visually on the number of mitoses seen per field. The distribution width, from the smoothed histogram as the DNA X square root area values beyond the maximum peak frequency, appeared to be larger in the high-grade malignant lymphomas, in accordance with their higher number of mitoses.     

An application of mathematical morphology to analysis of the size and shape of nuclei in tissue sections of non-Hodgkin's lymphoma

Using principles from the theory of mathematical morphology, a semiautomatic analysis of the size and shape of cell nuclei on tissue sections was carried out on a Leitz Texture Analysis System (Leitz-TAS). The four parameters proposed here are more discriminatory than conventional shape evaluation by the form factor (FF), which is based on the ratio of perimeter squared to area. The parameters quantified, respectively, nuclear elongation (ND), narrow (R1) and wide (R2) irregularities, and the distribution of R1 and R2 along the nuclear contour (ID). The properties of these parameters were tested nucleus-by-nucleus on 24 nuclear models. The methodology was then illustrated by a study of lymph node nuclei in non-Hodgkin's lymphoma (NHL). Prior to analysis, 45 lymphomas were classified into five categories of nuclear size and shape according to the International Working Formulation (IWF). Two hundred nuclei were measured on each lymph node section. Statistical interpretation was based upon an analysis of the nuclear surface area on sections and upon the mean values of R1, R2, and ND, the standard deviations of R1 and R2, and the percentage of cleaved nuclei detected by ID. The mean value of R2 discriminated best between the two sets of populations with regular and irregular nuclear contours, respectively. Parameters R1, ND, and ID permitted the distinction of certain NHL cases among populations with irregular nuclei. Nuclear invaginations decreased in depth as the nuclear area became greater. The median surface area was well correlated to the IWF, and the skewness coefficient (third statistical moment of the nuclear surface area distribution) was related to the number of nuclear size or shape subpopulations.     

Cytometric and histologic predictors of prognosis in ampullary carcinoma treated with pancreatico-duodenectomy

The association between several clinical, histologic and karyometric variables and the survival rates of patients with ampullary carcinoma was examined. Cases were limited to those treated exclusively by pancreaticoduodenectomy for which follow-up data was available, eliminating those cases with deaths due to other causes. The histologic type was classified as papillary, intestinal or mixed while the differentiation was recorded as well, moderate or poor. The stroma was categorized as scanty, moderate or abundant, and the tumor stage was evaluated according to Martin's classification. High-resolution morphometric and microphotometric (DNA content) evaluation of Feulgen-stained nuclei was performed using the microTICAS system. Statistical analyses were performed to examine the relationship between these variables and survival. Neither the tumor stage nor the presence of positive lymph nodes was a significant prognostic indicator, nor was the degree of differentiation or the amount of stroma. However, the survival showed a significant association with several karyometric variables and with the histologic type. Specifically, aneuploid DNA ploidy profiles, higher mean ploidy values and larger nuclei were associated with a lower survival rate. Short-term survivors (less than five years) had a mean ploidy of 2.8N, a mean 5N exceeding rate of 8.3% and a mean nuclear area of 41 sq microns, while long-term survivors (greater than or equal to five years) had corresponding means of 1.9N, 0.6% and 26 sq microns. These differences are all significant at a two-tailed significance level of less than .05 using a separate variance estimate t-test. In addition, papillary tumors showed a better prognosis than did intestinal or mixed tumors (Breslow P less than .04 and Mantel-Cox P less than .009).     

Cytophotometric and electron microscopic studies of chronic human skin lymphomas. II. Research on lymph node involvement

Results of electron microscopic and cytophotometric studies of biopsy material from lymph nodes of patients with cutaneous lymphomas with low degree of malignancy are discussed, with special reference to early diagnostic criteria of the disease. Specific characteristics of tumor tissue involve the presence of atypical lymphocytes with marginal condensed chromatin extrusions of nuclear material, deep invaginations of the nuclear membrane, nuclear pockets, excess of the mean DNA content per nucleus above the diploid standard level, more than 5% of nuclei being in the hyperdiploid area. Electron microscopic and cytophotometric methods allowed to diagnose the tumor injury of lymph nodes, when the traditional histological techniques revealed no signs of malignancy.     

Ultrastructure of adult T-cell leukemia/lymphoma

Eighteen cases of adult T-cell leukemia/lymphoma (ATLL) in Japan were analyzed by electron microscopy and compared with 5 cases of B-lymphoma and well-established groups of T-lymphomas (4 cases of T-lymphoblastic lymphoma and 2 cases of Sézary syndrome). Five hundred cells in each case, categorized ultrastructurally as to the cellular size and nuclear shape, showed an essentially pleomorphic cellular distribution in ATLL in remarkable contrast with the monomorphism of B-"histiocytic", B-well-differentiated lymphocytic, and T-lymphoblastic lymphomas. Some B-lymphomas and Sézary syndrome also showed pleomorphism. Cases of ATLL were classified according to the predominant cells and degree of nuclear irregularity, which delineated a spectrum of node-based, peripheral T-cells neoplasms probably encompassing T-immunoblastic sarcoma, T-zone lymphoma, as well as multilobated T-cell lymphoma. The characteristic fine structure of ATLL, in comparison with that of B-lymphomas, included slight to marked nuclear irregularity with convoluted-shape predominance, a specked chromatin pattern of the large cells, prominent lysosomes, and glycogen accumulation in addition to the difference in cellular distribution. Although T-lymphoblastic lymphoma and Sézary syndrome shared some of these features, the ultrastructural differentiation of ATLL from them seems to be possible.     

Comparative morphometric analysis of thyrocytes in a primary tumor and regional metastases in papillary thyroid gland cancer

OBJECTIVE: To perform a comparative analysis of thyrocyte nuclei and the aggregability degree in tumor cells in the thyroid gland with papillary cancer and in cervical lymph nodes with metastases. STUDY DESIGN: Thyrocytes in papillary cancer and those of cervical lymph nodes with its metastasis have been studied with the help of morphometry. RESULTS: As a result of mathematical transformation of the initial morphometric database, quantitative features of thyrocyte nuclei and aggregates characterizing the norm, papillary cancer and regional metastases were obtained. These features were united into 2 systems of decision criteria on the base of a set of quantitative features of thyrocyte nuclei and aggregates. Analysis was performed among the groups of comparison with the help of these systems. CONCLUSION: Malignant cells of the primary tumor have been shown to have evident quantitative features of nuclear atypia and a higher degree of aggregability compared with the norm. The differences between the quantitative features of nuclei and aggregates in malignant cells of the primary tumor in the thyroid gland and its metastases in cervical lymph nodes were less pronounced.     

Comparative study of the changes in microanatomical organization of lymph nodes located and functioning in the area of venous congestion

By the morphometric method the microanatomical organization of the popliteal and kidney lymph nodes of the type Vi was studied in venous congestion. In the first experimental series (lymph nodes localize in venous congestion area) the venous congestion in the popliteal lymph nodes was created by the ligature of caudal vena cava. In the second experimental series (lymph nodes, which clean the venous congestion area--the kidney vena was tied up. The small and short time increase of the marginal sinus volume and hypertrophy of the medullary cords, which seemed about medullary substance B-zone stimulation were characteristically for the lymph nodes, localize in the venous congestion area. The sharp increase of the marginal sinus volume during the long experimental time and paracortical T-zone hyperplasia were characteristically for the lymph nodes, which clean the venous congestion area.     

Morphometric study of the interphase nucleus in some radiosensitive and radioresistant mammalian cells

The radiosensitive cell populations, such as resting lymphocytes from thymus, spleen, lymph node and blood, have much smaller nuclei (Vn (nuclear volume) approximately 20 to 70 microns3) compared to radioresistant G0 cells from non-lymphoid tissues (liver, kidney, brain, heart; Vn approximately 75 to 2700 microns3). It is suggested that radiation-induced disorganization of nuclear structures and cell pycnosis (interphase death) are promoted in G0 lymphocytes because in normal physiological conditions their nuclei assume a higher degree of chromatin condensation. In contrast, dispersion of chromatin into larger nuclear volumes, such as those of most non-lymphoid G0 cells, may hinder or delay radiation-induced cell death.     

Pleomorphism of the nuclear envelope in breast cancer: a new approach to an old problem

In routine practice, nuclear pleomorphism of tumours is assessed by haematoxylin staining of the membrane-bound heterochromatin. However, decoration of the nuclear envelope (NE) through the immunofluorescence staining of NE proteins such as lamin B and emerin can provide a more objective appreciation of the nuclear shape. In breast cancer, nuclear pleomorphism is one of the least reproducible parameters to score histological grade, thus we sought to use NE proteins to improve the reproducibility of nuclear grading. First, immuno-fluorescence staining of NE as well as confocal microscopy and three-dimensional reconstruction of nuclei in cultured cells showed a smooth and uniform NE of normal breast epithelium in contrast to an irregular foldings of the membrane and the presence of deep invaginations leading to the formation of an intranuclear scaffold of NE-bound tubules in breast cancer cells. Following the above methods and criteria, we recorded the degree of NE pleomorphism (NEP) in a series of 273 invasive breast cancers tested by immunofluorescence. A uniform nuclear shape with few irregularities (low NEP) was observed in 135 cases or, alternatively, marked folds of the NE and an intranuclear tubular scaffold (high NEP cases) were observed in 138 cases. The latter features were significantly correlated (P-value <0.002) with lymph node metastases in 54 histological grade 1 and in 173 cancers with low mitotic count. Decoration of the NE might thus be regarded as a novel diagnostic parameter to define the grade of malignancy, which parallels and enhances that provided by routine histological procedures.     

Nuclear morphometric measurements in rectal adenocarcinoma cells of patients of different races

The reasons for the different long-term prognoses of black and white patients following curative resection of a rectal adenocarcinoma are unknown. In order to investigate whether rectal adenocarcinomas in blacks have clinical or pathologic characteristics that are different from rectal adenocarcinomas in whites, 149 patients with potentially curable rectal cancers resected at the University of Chicago Medical Center between 1965 and 1981 were retrospectively analyzed. Clinical records, pathology reports and pertinent slides were reviewed in each case. In 142 cases, enough histologic material was available to perform nuclear photometric measurements and determinations of DNA content by the slide-cytophotometric method. There was no difference between black and white patients in the stage, differentiation degree, morphology and ploidy of the tumors, or in the presence of microinvasion, metastases and mucin production. However, adenocarcinoma cells of black patients had smaller nuclei than did the corresponding nuclei of white patients (54.7 +/- 2.34 sq microns versus 58.9 +/- 1.84 sq microns; P less than .05), and the neoplastic nuclei of black patients were significantly rounder and more regular than the nuclei of white patients (mean roundness factors of 1.1 +/- 0.003 vs. 1.11 +/- 0.005; P less than .05). Although these findings will require confirmation from other large clinical series, they suggest that the different prognoses of black and white patients after curative resection of a rectal adenocarcinoma may be explained by a different tumor behavior intrinsically related to different karyotypic characteristics of the neoplastic cells.     

Distribution of dendritic reticulum cells in follicular lymphoma and reactive hyperplasia. Light microscopic identification and general morphology

Reactive and neoplastic follicles in lymph nodes showing changes of (1) follicular hyperplasia and (2) follicular lymphoma were examined for the presence of dendritic reticulum cells (DRC). These cells could be identified under the light microscope after comparing electron microscopical sections with subsequent 1 micro sections of reactive germinal centres. Quantitative light microscopical evaluation showed that DRC, both mononuclear and binuclear forms, were less numerous in neoplastic follicular structures than in reactive follicles. Before determining the frequency of binucleated DRC in follicular tissue their morphology was studied first in cell suspensions. DRC isolated by enzymatic treatment of tonsils and reactive lymph nodes were morphologically identical and contained one, or at most two, nuclei arranged in a typical doublet formation. Stereological calculations - made on three dimensional models of nuclear complexes prepared from serial tissue sections - indicated that 51 to 68% of DRC in reactive germinal centres were binucleated, whereas in neoplastic follicles this figure is 18 to 23%. The multinucleated giant cell forms of DRC described by others result from complex formation with other DRC or lymphoid cells. The smaller number of DRC and the lower frequency of binucleated DRC in follicular lymphomas suggest that differentiation of DRC from stromal cells is less complete in these neoplasms. The ability to identify DRC reliably by light microscopy offers a new means to define the difference in frequency of DRC. This may be of practical value in distinguishing reactive germinal centres from neoplastic follicles.     

Density of intranodal lymphatics and VEGF-C expression in B-cell lymphoma and reactive lymph nodes

Lymphatic vasculature in solid tumors may serve as the pathway for metastatic spread of the cancer to the regional lymph nodes and to distant organs. Controversy still exists whether tumors metastasize through existing lymphatics or through newly formed vessels (lymphangiogenesis). The role of lymphangiogenesis in lymphoma spread and proliferation is not clearly established. VEGF-C is the most potent inducer of lymphangiogenesis. LYVE-1 was shown to be a specific marker for lymphatic vessels in normal and tumor tissue. The aim of the present study was the evaluation of lymph node LYVE-1-positive lymphatic sinus density (LSD) and VEGF-C expression in patients with non-Hodgkin's lymphoma (nHL) and in reactive lymph nodes. Sixty paraffin-embedded lymph nodes from newly diagnosed patients with B-cell nHL were evaluated. Twelve lymph node biopsy specimens from adult patients with reactive lymphonodulitis were used as controls. Sections of lymph nodes were stained immunohistochemically for LYVE-1 and VEGF-C. VEGF-C expression in lymph nodes of nHL patients was low and not significantly different from that in the control (p = 0.6). Moreover, VEGF-C expression did not differ significantly between aggressive and indolent lymphomas (p = 0.53). Similarly we did not find differences in LSD in aggressive nHL and in indolent nHL (p=0.49). The mean LSD in reactive lymph nodes was higher than in nHL (p = 0.03). Only in 2 out of 12 reactive lymph nodes LYVE-1-positive vessels were absent. In all groups we demonstrated a strong positive correlation between VEGF-C and LYVE-1-expression (p = 0.0001). Higher LSD in reactive lymph nodes as compared to those of nHL patients suggests that lymphoma proliferation leads to the destruction of the existing lymphatics rather than to lymphangiogenesis within lymph nodes. NHL are not associated with increased expression of VEGF-C nor increased LYVE-1-positive lymphatic sinuses density within lymph nodes.     

Morphometry of nuclei of the normal and malignant prostate in relation to DNA ploidy.

Fine needle biopsies from 70 patients with prostate carcinoma and 10 patients with benign hyperplasia were used to study area, variation in size and form factors of the nuclei by image analysis. The results were related to DNA ploidy of the cell populations as measured by flow cytometry, cytologic grade and patients' survival. Nuclear area differed significantly between benign lesions and tumors. It increased in diploid low-grade tumors from a normal value of 54.2 +/- 3.1 microns2 to 75.6 +/- 5.3 microns2. In aneuploid tumors with an increase in the chromosome number, the nuclear size further increased to about twice that of benign nuclei. Variation in size also differed between benign and malignant epithelium, with a further increase between diploid and gross aneuploid tumors. While nuclear size and variation in nuclear size made it possible to discriminate malignant from benign lesions, form factor did not differ between benign and malignant lesions. In follow-up, however, none of these factors reached significance for predicting survival.     

Morphometric distinction of granulomas in tuberculosis and sarcoidosis. Difference in nuclear profiles

Morphometric measurements were carried out on epithelioid-cell nuclei of noncaseating granulomas in paraffin-embedded sections of lymph node biopsy specimens originating from 15 patients with sarcoidosis and from 18 patients with tuberculosis. The results, which were obtained with the help of a computer-assisted tracing device, established highly significant differences in shape and size of epithelioid-cell nuclear profiles in these two diseases. Direct measurements and computations on these results showed that epithelioid-cell nuclear profiles in sarcoidosis have a smaller mean and median perimeter (boundary length), area and long diameter (maximum length of the nuclear profile) and exhibit a more plump, elliptical-like shape than do those in tuberculous granulomas. Epithelioid-cell nuclear profiles of the latter, as a whole, show more irregular contours, resembling those, e.g., of the sole of a shoe or other elongated patterns. Differences in shape of nuclear profiles were best demonstrated by a size-independent form factor (4 pi *area/perimeter2).     

Fine needle aspiration biopsy of the breast. Value of nuclear morphometry after different sampling methods

OBJECTIVE: To study the potential of nuclear morphometry in supporting the interpretation of fine needle aspiration biopsy (FNAB) samples of the breast fixed in 50% ethanol and centrifuged on slides. STUDY DESIGN: Computerized morphometry was used to outline the nuclei of breast epithelial cells in breast cancer, fibroadenoma and fibrocystic disease. The diagnoses were histologically confirmed. We applied 2 different sampling methods (measurements done on cell groups and on free cells). RESULTS: The mean nuclear area of cell groups of malignant samples (23) varied from 42 to 125 microns 2, in fibroadenomas from 30 to 50 microns 2 and in fibrocystic disease from 26 to 57 microns 2. The mean nuclear area of free cells varied as follows: cancer, 66-181 microns 2; fibroadenoma, 33-70 microns 2; fibrocystic disease, 35-60 microns 2. Apocrine metaplasia was excluded from comparison on a morphologic basis. CONCLUSION: The study suggests that if the mean nuclear area of cell groups is < 42 microns 2, the lesion is probably benign; if > 57 microns 2, and apocrine metaplasia is excluded, malignancy should be considered. The differential diagnosis between carcinoma and fibroadenoma could be based on free cells: mean area of free cell nuclei < or = 65 microns 2 suggested a benign lesion, and of > or = 71 microns 2 suggested a malignant lesion. Morphometric nuclear size features (exemplified by nuclear area) appeared efficient in distinguishing between malignant and benign lesions when measured from free cells and cell groups.     

Proinflammatory chemokine gene expression influences survival of patients with non-Hodgkin's lymphoma

The migration, survival and proliferation of cells is the basis for all physiologic and pathologic processes in the human body. All these reactions are regulated by a complex chemokine network that guides lymphocytes homing, chemotaxis, adhesion and interplay between immunologic system response cells. Chemokines are also responsible for metastatic dissemination of cancers, including Hodgkin's and non-Hodgkin's lymphomas. The purpose of this study was to determine chemokine gene expression (CXCL8, CXCL10, CCL2, CCL3, CCL4 and CCL5) in lymphoma lymph nodes compared to their expression in reactive lymph nodes. We also analyzed the influence of chemokine gene expression on the survival of lymphoma patients. Chemokine gene expression was evaluated in 37 lymphoma lymph nodes and in 25 samples of reactive lymph nodes. Gene expression of chemokines CXCL8, CXCL10, CCL2, CCL3, CCL4 and CCL5 was measured using the PCR method. Statistical analysis was performed using CSS Statistica for Windows (version 7.0) software. Probability values 〈 〈 0.05 were considered statistically significant and those between 0.05 and 0.1 as indicative of a trend. We found lower CXCL8 and CXCL10 gene expression in lymphoma lymph nodes compared to reactive lymph nodes. In the cases of CCL2 and CCL3, expression in lymphomas was higher than in reactive lymph nodes. Patients with high expression of CCL2 and CXCL10 had shorter survival.     

Sperm nuclear transformations consist of enlargement and condensation coordinate with stages of meiotic maturation in fertilized Spisula solidissima oocytes

Rates of sperm nuclear expansion were measured and correlated with processing of the maternal chromatin in synchronous populations of fertilized surf clam (Spisula solidissima) oocytes fixed at regular intervals following insemination and stained with the DNA fluorochrome Hoechst 33342. Sperm nuclei expanded in four distinct phases each temporally coordinate with events of meiotic maturation: germinal vesicle stage (phase A), germinal vesicle breakdown (phase B), polar body formation (phase C), and female pronuclear development (phase D). Sperm nuclei were essentially unchanged during phase A (rate = 0.1 micron2/min, enlarged during phases B (rate = 8.2 microns2/min) and D (rate = 6.2 microns2/min), and condensed during phase C (rate = -1.9 micron2/min). Sperm nuclear enlargement during phase D was significantly less in polyspermic and polygynic zygotes. The effects of various treatments (temperature, microtubule disruption, pH alterations, and metabolic and protein synthesis inhibitions) which perturbed sperm nuclear enlargement and meiotic processing of the maternal chromatin indicated that the two processes are coupled and may be linked by common regulatory agents.