Model-Based Assessment of the Role of Uneven Partitioning of Molecular Content on Heterogeneity and Regulation of Differentiation in CD8 T-Cell Immune Responses.

Activation of naive CD8 T-cells can lead to the generation of multiple effector and memory subsets. Multiple parameters associated with activation conditions are involved in generating this diversity that is associated with heterogeneous molecular contents of activated cells. Although naive cell polarisation upon antigenic stimulation and the resulting asymmetric division are known to be a major source of heterogeneity and cell fate regulation, the consequences of stochastic uneven partitioning of molecular content upon subsequent divisions remain unclear yet. Here we aim at studying the impact of uneven partitioning on molecular-content heterogeneity and then on the immune response dynamics at the cellular level. To do so, we introduce a multiscale mathematical model of the CD8 T-cell immune response in the lymph node. In the model, cells are described as agents evolving and interacting in a 2D environment while a set of differential equations, embedded in each cell, models the regulation of intra and extracellular proteins involved in cell differentiation. Based on the analysis of in silico data at the single cell level, we show that immune response dynamics can be explained by the molecular-content heterogeneity generated by uneven partitioning at cell division. In particular, uneven partitioning acts as a regulator of cell differentiation and induces the emergence of two coexisting sub-populations of cells exhibiting antagonistic fates. We show that the degree of unevenness of molecular partitioning, along all cell divisions, affects the outcome of the immune response and can promote the generation of memory cells.

Nasty viruses, costly plasmids, population dynamics, and the conditions for establishing and maintaining CRISPR-mediated adaptive immunity in bacteria.

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) abound in the genomes of almost all archaebacteria and nearly half the eubacteria sequenced. Through a genetic interference mechanism, bacteria with CRISPR regions carrying copies of the DNA of previously encountered phage and plasmids abort the replication of phage and plasmids with these sequences. Thus it would seem that protection against infecting phage and plasmids is the selection pressure responsible for establishing and maintaining CRISPR in bacterial populations. But is it? To address this question and provide a framework and hypotheses for the experimental study of the ecology and evolution of CRISPR, I use mathematical models of the population dynamics of CRISPR-encoding bacteria with lytic phage and conjugative plasmids. The results of the numerical (computer simulation) analysis of the properties of these models with parameters in the ranges estimated for Escherichia coli and its phage and conjugative plasmids indicate: (1) In the presence of lytic phage there are broad conditions where bacteria with CRISPR-mediated immunity will have an advantage in competition with non-CRISPR bacteria with otherwise higher Malthusian fitness. (2) These conditions for the existence of CRISPR are narrower when there is envelope resistance to the phage. (3) While there are situations where CRISPR-mediated immunity can provide bacteria an advantage in competition with higher Malthusian fitness bacteria bearing deleterious conjugative plasmids, the conditions for this to obtain are relatively narrow and the intensity of selection favoring CRISPR weak. The parameters of these models can be independently estimated, the assumption behind their construction validated, and the hypotheses generated from the analysis of their properties tested in experimental populations of bacteria with lytic phage and conjugative plasmids. I suggest protocols for estimating these parameters and outline the design of experiments to evaluate the validity of these models and test these hypotheses.

Comprehensive assessment and mathematical modeling of T cell population dynamics and homeostasis.

Our current view of T cell differentiation and population dynamics is assembled from pieces of data obtained from separate experimental systems and is thus patchy. We reassessed homeostasis and dynamics of T cells 1) by generating a mathematical model describing the spatiotemporal features of T cell differentiation, and 2) by fitting this model to experimental data generated by disturbing T cell differentiation through transient depletion of dividing T cells in mice. This specific depletion was obtained by administration of ganciclovir to mice expressing the conditional thymidine kinase suicide gene in T cells. With this experimental approach, we could derive quantitative parameters describing the cell fluxes, residence times, and rates of import, export, proliferation, and death across cell compartments for thymocytes and recent thymic emigrants (RTEs). Among other parameters, we show that 93% of thymocytes produced before single-positive stages are eliminated through the selection process. Then, a postselection peripheral expansion of naive T cells contributes three times more to naive T cell production than the thymus, with half of the naive T cells consisting of dividing RTEs. Altogether, this work provides a quantitative population dynamical framework of thymocyte development, RTEs, and naive T cells.

Assessment of the cell-mediated immune response in chickens by detection of chicken interferon-gamma in response to mitogen and recall Newcastle disease viral antigen stimulation.

The potential of a capture enzyme-linked immunosorbent assay (ELISA) specific for chicken interferon-gamma (ChIFN-gamma) has been evaluated as a tool to assess cell-mediated immunity (CMI) in the chicken. In a first step, ChIFN-gamma production and cell proliferation of mitogen-activated chicken splenocytes have been compared. In general, for each of the stimulation conditions where significant proliferation was observed, production of ChIFN-gamma could be measured by ELISA. In our hands, the combination of ionomycin and phorbol-12-myristate 13-acetate or the use of recombinant chicken interleukin-2 gave the most satisfactory results. Then, the CMI response induced by live or killed Newcastle disease virus (NDV) vaccines has been evaluated sequentially by ex vivo antigen-specific ChIFN-gamma production and cell proliferation of splenocytes from immune chickens. The ex vivo data showed that both types of NDV vaccines are capable of stimulating CMI responses to NDV in chickens as measured by the ChIFN-gamma ELISA. However, most of the chickens vaccinated with the live vaccine produced ChIFN-gamma after antigen recall stimulation, from 2 to 4 weeks after vaccination, when only some chickens vaccinated with the inactivated vaccine showed a specific response 4 weeks after vaccination. No significant proliferative responses to either NDV vaccine were detectable during the 4 weeks of the study. From our results, it appears that antigen-specific ChIFN-gamma production can be used as a good indicator of actively acquired immunity to NDV and that the sensitivity range of the capture ELISA test is well adequate to measure ex vivo release of ChIFN-gamma.

Biochemical and functional assessment of equine lymphocyte phosphodiesterases and protein kinase C.

Lymphocytes play an important role in allergic inflammation and have been implicated in the pathogenesis of equine allergic skin and respiratory disease. Targeting intracellular signalling pathways in human lymphocytes has demonstrated a role for both phosphodiesterase and protein kinase C in cell activation. The aim of this study was to measure total cyclic nucleotide hydrolysing phosphodiesterase activity and to identify the phosphodiesterase and protein kinase C isoenzymes present in equine lymphocytes. The functional significance of these isoenzymes was then investigated by examining their role in peripheral blood mononuclear cell proliferation using isoenzyme selective inhibitors. Total cyclic adenosine monophosphate hydrolysing phosphodiesterase activity was double that of cyclic guanosine monophosphate (30+/-2 pmol/min mg versus 16+/-3 pmol/min mg for cyclic adenosine and cyclic guanosine monophosphate phosphodiesterase activity, respectively). Evidence for the presence of PDE1, 3, 4 and 5 was obtained and PKCalpha, beta, delta, eta, iota, theta and zeta were identified. Selective inhibitors of PDE4, PKCdelta and conventional PKCs alpha and beta caused significant inhibition of mitogen-induced peripheral blood mononuclear cell proliferation. This study demonstrates a functional role for specific signalling isoenzymes and suggests that, in the context of allergic inflammation, targeting inflammatory cells involved in disease pathogenesis with relevant isoenzyme inhibitors may have therapeutic potential.

Estimating hypermutation rates from clonal tree data.

To understand the mechanisms underlying the varying patterns of mutations that occur during immune and autoimmune responses, estimates of the somatic hypermutation rate are critical. However, despite its significance, precise estimates of the mutation rate do not currently exist. Microdissection studies of mutating B cell clones provide an opportunity to measure this rate more accurately than previously possible. Each microdissection provides a number of clonally related sequences that, through the analysis of shared mutations, can be genealogically related to each other. The shape of these clonal trees is influenced by many processes, including the hypermutation rate. We have developed two different methods to estimate the mutation rate based on these data. These methods are applied to two sets of experimental data, one from an autoimmune response and one from the antihapten response to (4-hydroxy-3-nitrophenyl)acetyl (NP). Comparable mutation rates are estimated for both responses, 0.7-0.9 x 10(-3) and 0.9-1.1 x 10(-3) bp(-1) division(-1) for the autoimmune and NP responses, respectively. In addition to comparing the results of the two procedures, we investigate the effect on our estimate of assumptions, such as the fraction of lethal mutations.

Models for antigen receptor gene rearrangement. III. Heavy and light chain allelic exclusion.

The extent of allelic exclusion in Ig genes is very high, although not absolute. Thus far, it has not been clearly established whether rapid selection of the developing B cell as soon as it has achieved the first productively rearranged, functional heavy chain is the only mechanism responsible for allelic exclusion. Our computational models of Ag receptor gene rearrangement in B lymphocytes are hereby extended to calculate the expected fractions of heavy chain allelically included newly generated B cells as a function of the probability of heavy chain pairing with the surrogate light chain, and the probability that the cell would test this pairing immediately after the first rearrangement. The expected fractions for most values of these probabilities significantly exceed the levels of allelic inclusion in peripheral B cells, implying that in most cases productive rearrangement and subsequent cell surface expression of one allele of the heavy chain gene probably leads to prevention of rearrangement completion on the other allele, and that additional mechanisms, such as peripheral selection disfavoring cells with two productively rearranged heavy chain genes, may also play a role. Furthermore, we revisit light chain allelic exclusion by utilizing the first (to our knowledge) computational model which addresses and enumerates B cells maturing with two productively rearranged kappa light chain genes. We show that, assuming that there are no selection mechanisms responsible for abolishing cells expressing two light chains, the repertoire of newly generated B lymphocytes exiting the bone marrow must contain a significant fraction of such kappa double-productive B cells.

Assessment of proliferation index with MIB-1 as a prognostic factor in radiation therapy for cervical cancer.

OBJECTIVES: The relationship between the expression of murine monoclonal antibody MIB-1, which reacts with Ki-67 nuclear antigen, a marker for proliferating cells, and the prognosis of stage IIIb cervical cancer after radiation therapy was analyzed. METHODS: A total of 67 patients with stage IIIb cervical cancer who had received radiation therapy were included in the retrospective study. The labeled streptavidin-biotin method was used for immunohistochemical staining of the MIB-1 protein. RESULTS: In 32 patients showing a high MIB-1 index (percentage of cells labeled with MIB-1 >/=26.4%), the cumulative 5- and 8-year survival rates were 75.8 and 61.5%, respectively, significantly better (P < 0.05) than those in 35 patients with a low MIB-1 index (<26.4%) (59.6 and 41.1%, respectively). Serum squamous cell carcinoma antigen levels, an index of the response to radiation therapy, decreased to

Functional assessment in vitro of human-complement-dependent antibody-induced cytotoxicity of neoplastic cells.

The complement system is one potential cytotoxic effector mechanism that might be effective in immunotherapy of cancer using monoclonal antibodies (mAb) directed against tumor antigens. In order to evaluate the treatment outcome from trials using mAb in cancer patients, assessment of complement-dependent cytotoxicity (CDC) may therefore be of interest. Here we describe the elaboration of a CDC assay in vitro using a rat hepatoma cell line, H4-II-E, as target cells sensitised with mAb F12, directed against the tumor-associated ganglioside antigen fucosyl-GMI. Sensitised cells were incubated with various concentrations of fresh serum as complement source for 48 h and cytotoxicity was then assessed by the tetrazolium bromide (MTT) test. A large variation in CDC efficacy was observed between individual serum donors. No differences in CDC could be seen between healthy donors and cancer patients. The CDC showed a strong correlation to the serum concentrations of complement factor C4, supporting the validity of the assay. Our results suggest that there may be significant variations in complement function within and between individuals that might influence the outcome of clinical mAb therapy. The H4/F12 CDC assay described here, together with measurement of individual complement factors. such as C4, should be further validated in cancer patients at various disease stages and phases of treatment.

Assessment of proliferative and colony-forming capacity after successive in vitro divisions of single human CD34+ cells initially isolated in G0.

Exit of primitive hematopoietic progenitor cells (HPCs) from the G0 phase of the cell cycle in response to in vitro cytokine stimulation is a limiting step in successful ex vivo expansion. Simultaneous DNA/RNA staining with Hoechst 33342 and pyronin Y was used to separate human bone marrow CD34+ cells residing in G0 (G0CD34+) from those cycling in G1 and S/G2+M. Compared with CD34+ cells isolated in G1, G0CD34+ cells were characterized by a delayed response to cytokine stimulation and were enriched for long-term hematopoietic culture-initiating cells. We next compared the activation kinetics of individually sorted G0CD34+ cells stimulated with stem cell factor (SCF), flt3-ligand (FL), or interleukin-3 (IL-3) as single factors. In a novel clonal proliferation assay, the functional status of cells that had remained quiescent after an initial 7-day period and of those that had completed successive division cycles under each of these three factors was evaluated by assessment of subsequent proliferative capacity and maintenance of colony-forming cell precursor (pre-CFC) activity. All three cytokines were equally able to support the survival of primitive HPCs in the absence of cell division. Cells that did not respond to any cytokine stimulation for 7 days retained higher proliferative and pre-CFC activities than dividing cells. The hematopoietic function of cells that divided in response to SCF, FL, or IL-3 decreased after each division cycle. However, G0CD34+ cells displayed a heterogeneous response pattern to cytokine stimulation whereby SCF appeared to have a superior ability to promote the cycling of cells with high proliferative and pre-CFC activities. These results indicate that HPCs reside in opposing hierarchies of hematopoietic potential and responsiveness to cytokine stimulation. The data also begin to indicate relationships between cellular division in response to different stimuli and maintenance of hematopoietic function.

In vivo and in vitro assessment of B-B cell interactions: inhibition of proliferation and antibody production of the CRIA B cells mediated by the surface immunoglobulins of anti-CRIA B cells.

In this work we have assessed the effect of cell surface anti-immunoglobulin (Ig) of anti-idiotypic B cells on their idiotypic counterparts in vivo and in vitro, as a surrogate for soluble anti-surface Ig, using the well-characterized anti-arsonate system. The response of A/J mice against the hapten arsonate coupled to keyhole limpet hemocyanin (ARS-KLH) is dominated by a closely related family of antibodies sharing the same determinant, named the CRIA idiotype. We show herein that a massive induction of anti-CRIA B cells, subsequent to immunization with the mAb 3665 (CRIA+, arsonate binding) coupled to KLH, mediated a strong and long-lasting inhibition of this dominant oligoclonal response to arsonate. The titer of anti-arsonate antibodies remained, however, unchanged. Adoptive transfers to x-irradiated syngeneic mice showed that anti-CRIA-producing B cells have a direct effect on induction of inhibition. This was supported by the in vitro data where irradiated anti-CRIA B cells could induce inhibition of both antibody production and mitogenesis of their counterparts, CRIA B cells. This inhibitory effect could be decreased when the surface anti-surface Ig were hidden by the 3665 Fab fragments but not by anti-MHC class II antibodies. These interactions between CRIA and anti-CRIA B cells were solely Igh restricted and the inhibition was likely initiated by hyperaggregation of surface Ig. The presence of ARS-KLH-primed T cells in vitro could prevent the growth inhibition but not the suppression of antibody production. A similar profile was noticed in vitro for soluble polyclonal rabbit anti-CRIA Ab. All together, our data suggest that a negative signaling in B cells may be initiated by surface Ig of their idiotypic partners subsequent to a strong cross-linking of their surface Ig receptors.

Nuclear morphometry, immunohistochemical staining with Ki-67 antibody and mitotic index in the assessment of proliferative activity and prognosis of primary malignant melanomas of the skin.

Nuclear morphometry, immunohistochemical staining with Ki-67 antibody and mitotic index were studied in primary cutaneous malignant melanomas. The number of Ki-67 positive cells/ 200 tumor cells did not correlate with any nuclear morphometrical parameters, and it only approached but did not reach significant correlation with melanoma thickness according to Breslow. The nuclear area, short axis and long axis correlated with melanoma thickness, but the nuclear axis ratio (which reflects the sphericity of nuclei) and melanoma thickness did not show significant correlation. Mitotic index was higher in thick melanomas and in melanomas with high Ki-67 positivity, large nuclear area, long nuclear short axis, and small nuclear axis ratio. In Cox's stepwise proportional hazard model, melanoma thickness and the nuclear axis ratio were significant independent prognostic factors for patient survival, while the nuclear area, short axis and long axis, gender, age, Clark level, mitotic index and Ki-67 positivity lacked significant independent prognostic value. The results suggest that the proliferative activity of tumor cells does not alone explain the great importance of tumor thickness as prognosticator in melanoma. The thickness of melanoma measured according to Breslow and the nuclear axis ratio are more efficient prognosticators in melanoma than parameters associated with proliferation.

Immunohistochemical assessment of proliferation rate of breast carcinoma cells using Ki-67, MIB-1 and anti-PCNA monoclonal antibodies.

MIB-1 Ki-67 and PCNA scores in infiltrating ductal NOS breast carcinomas were compared. The correlation between MIB-1, Ki-67 and PCNA indices and several clinicopathological factors that have prognostic significance in breast cancer was also assessed. The mean Ki-67, MIB-1 and PCNA indices were 13.4%, 19.4%, 27.6%, respectively. Significant positive linear correlation was found only between Ki-67 and MIB-1 indices. PCNA score did not correlate with Ki-67 and MIB-1 indices. The significant correlation between Ki-67 and MIB-1 scores and histological grade was found. There was no correlation between Ki-67 and MIB-1 indices and axillary lymph node status or tumor diameter. The results suggest that MIB-1 antibody is an excellent tool for assessment of proliferative rate of breast cancer cells in paraffin sections.

Assessment of cell proliferation in normal and pathological bone marrow biopsies: a study using double sequential immunophenotyping on paraffin sections.

The proliferative activity of the haematopoietic and plasma cells in bone marrow was evaluated under normal and neoplastic conditions, by means of a sequential double immunostaining technique, using monoclonal antibody MIB-1 recognizing the cell proliferation-associated nuclear antigen Ki-67, and antibodies against glycophorin-C, myeloperoxidase, factor VIII-related antigen, and immunoglobulin light chains. Fifty-eight B5 fixed, paraffin-embedded bone marrow biopsies were analysed, including 11 normal controls. 10 cases of myelodysplasia, 14 cases of chronic myeloproliferative disorder, eight cases of acute non-lymphoid leukaemia, and 15 cases of myeloma. In normal marrows, the highest proliferative activity was noticed in the erythroid cells (75% to 95%; mean 90%), in comparison with myeloid precursors (15% to 80%; mean 38%), and megakaryocytes (10% to 20%; mean 14%): no Ki-67 positive plasma cells were found. In all investigated haematological disorders, the expression of MIB-1 by erythroid cells was similar to that observed in controls. Similarly, the percentage of MIB-1 + myeloid precursors in chronic myeloproliferative disorders and myelodysplasia largely overlapped the values observed in normals, and comparable values were also found in the blast cells from acute non-lymphoid leukaemia type M1 and M2. These findings suggest that the evaluation of either erythroid or myeloid proliferative activity is of little value in the differential diagnosis between these myeloproliferative disorders. By contrast, the obvious increase of Ki-67 expression of megakaryocytes in chronic myeloproliferative disorders, with labelling also of micro-megakaryocytes, might sustain the diagnosis in controversial cases. Since cases of mature myeloma showed less than 2% of Ki-67 positive cells, evaluation of proliferative activity is of no value in the differential diagnosis with reactive plasmacytosis. The sequential double immunophenotyping for Ki-67 antigen and for haematopoietic cell lineage-associated markers can be applied in a consistent manner to routine bone marrow biopsies to evaluate proliferating cells in normal and neoplastic conditions.

Assessment of the therapeutic potential of cytokines, cytotoxic drugs and effector cell populations for the treatment of multiple myeloma using the 5T33 murine myeloma model.

The therapeutic potential of six cytokines, eight cytotoxic drugs and two effector cell populations for the treatment of multiple myeloma was assessed in vitro using the 5T33 murine myeloma model. The efficacy of combination IFN-alpha and melphalan therapy was also evaluated in vitro and in vivo. Of the cytokines tested in vitro using the MTT assay, only IFN-alpha demonstrated significant inhibition of myeloma cell growth at non-toxic concentrations (ED50 = 1508.3 +/- 181.3 U/mL and 2617.9 +/- 334.0 U/mL for murine IFN-alpha [mIFN-alpha] and human IFN-alpha hybrid B/D [hIFN-alpha B/D], respectively). The ED50 for the eight cytotoxic drugs tested ranged from 2.3 x 10(-9) to 4.3 x 10(-13) mol/L and all were within the therapeutic range for humans. Combination hIFN-alpha B/D and melphalan were found to be additive in their inhibitory effects on myeloma cell growth in vitro and this finding was confirmed in vivo in C57BL/KaLwRij mice bearing disseminated 5T33 myeloma. Control animals demonstrated a median survival duration of 25.3 days whereas hIFN-alpha B/D or melphalan treatment alone increased survival to 30.5 and 33.3 days, respectively (P < 0.001). Combination IFN-alpha/melphalan therapy increased median survival duration to 38.5 days (P < 0.001) which was also significantly greater than that obtained with single agent therapy (P < 0.01). The murine myeloma cells were found to be resistant to NK cell lysis but susceptible to lysis by LAK cells (49.3 +/- 6.3% lysis at an effector to target ratio of 100:1).(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of cell proliferation in hydatidiform mole using monoclonal antibody MIB1 to Ki-67 antigen.

AIMS: To assess the role of Ki67 immunoreactivity in predicting the clinical progress of hydatidiform mole. METHODS: Tissue from 87 hydatidiform moles, 11 normal first trimester placentas, 11 normal term placentas and 17 spontaneous abortions were examined for expression of Ki67 antigen, using the monoclonal antibody MIB1. RESULTS: Ki67 immunoreactivity was significantly higher in the tissue from normal first trimester placentas than in that from normal term placentas and spontaneous abortions. Among the 87 patients with hydatidiform moles studied, 20 developed persistent gestational trophoblastic disease and required subsequent treatment. There was no statistically significant difference in the Ki67 index between the 20 patients who developed persistent disease and those who did not. CONCLUSION: Hydatidiform moles which give rise to persistent trophoblastic disease do not have a higher proliferative rate than those which do not. The Ki67 index is not useful for predicting the prognosis of molar pregnancies.

Comparison of villous trophoblast proliferation rate in hydatidiform mole and non-molar abortion by assessment of proliferating cell nuclear antigen expression.

This study examines the proliferative activity of trophoblast in hydatidiform mole, non-molar hydropic abortion and non-molar spontaneous abortion. Nine cases of complete mole, 10 cases of partial mole, eight cases of non-molar hydropic abortion and six cases of non-hydropic second trimester abortion were examined by routine histopathology and the rate of cell proliferation was assessed by immunoreactivity for proliferating cell nuclear antigen (PCNA). Hydropic abortion showed a significantly lower PCNA index than complete mole and partial mole. There was no significant difference in PCNA index between partial mole and non-hydropic abortion. The trophoblast of partial hydatidiform mole demonstrates significant cell proliferation but this, although higher than that of hydropic abortion, is no higher than that of non-hydropic abortion of a similar gestational age. The role of partial mole as a precursor of persistent gestational trophoblastis disease remains unclear.

[Early or late orchidopexy? An evaluation of germ cell proliferation by PCNA immune expression]. / Orquidopexia precoz o tardía? Valoración de la proliferación de las células germinales mediante inmunoexpresión de PCNA.

A immunocytochemical study for detection of proliferating cell nuclear antigen (PCNA) in order to quantify the number of PCNA-positive spermatogonia, and cytophotometric determination of spermatogonial DNA were performed in cryptorchid and control testes. The number of PCNA-positive spermatogonia, and the average DNA content of spermatogonia in the cryptorchid testes were altered from first years of age. These precocious spermatogonial alterations suggest that the early surgical testicular descent doesn't prevent lesions of germ cells.

Assessment of proliferative activity in carcinomas of the human alimentary tract by Ki-67 immunostaining.

The monoclonal antibody (MAb) Ki-67, which is directed against a proliferation-associated nuclear antigen, was used to measure tumor proliferation in 165 carcinomas of the esophagus, stomach, colon and rectum with an indirect immunoperoxidase technique. The percentage of Ki-67-positive tumor cells (Ki-67 index) was evaluated with the point-counting method. The Ki-67 index in gastric cancers (mean, 24.8%; standard deviation, 11.1%) was significantly lower than in tumors of the esophagus (35.7 +/- 12.6%), colon (37.6 +/- 15.2%), and rectum (34.3 +/- 16.4%). A wide range of the Ki-67 index (5.9-75.3%) could be observed within the various tumor types. In recurrent colorectal carcinomas, the Ki-67 index significantly increased to 51.9%. The Ki-67 index was independent of pathologic (e.g., TNM-stage, grading, tumor volume, tumor site) and clinical variables (age and gender of the patients). A marked heterogeneity of Ki-67 expression within different tumor stages was noted. Statistically significant regional variations in tumor proliferation existed between different areas within the same tumor.