Cytokine effects on cell viability and death of prostate carcinoma cells.

We analyzed the effects of IL-13, IFN- γ , and IL-1 ß on cell viability and death of LNCaP and PC-3 cells and major signaling pathways involved in these effects. Significant increase of LNCaP cell death (apoptotic and necrotic) and increased levels of active caspase 3 were observed in cells treated with inhibitors of ERK 1/2 (UO126) and p38 (SB203580) prior to IL-1 ß treatment in comparison to cells treated with UO126, SB203580, or IL-1 ß alone. Significant increase of LNCaP but not PC-3 cell death was detected after treatment with LY-294002 (inhibitor of phosphatidylinositol 3-kinase). No significant increase of LNCaP and PC-3 cell death was observed after treatment with SP600125 (inhibitor of JNK), SB203580 (inhibitor of p38), UO126 (inhibitor of ERK 1/2), or BAY 11-7082 (inhibitor of NF- κ B). Reduced c-FLIPL expression was observed in LNCaP cells treated with LY-294002. The significant potentiation of LNCaP cell death by inhibition of ERK 1/2, p38, and PI3-K pathways may provide a rationale for therapeutic approach in androgen-dependent prostate cancer.

EGFR gene gain and PTEN protein expression are favorable prognostic factors in patients with KRAS wild-type metastatic colorectal cancer treated with cetuximab.

INTRODUCTION: Cetuximab is a monoclonal epidermal growth factor receptor (EGFR)-targeting antibody, used in the treatment of colon cancer. KRAS mutation status is strongly predictive of cetuximab efficacy, but more predictive factors are needed for better patient selection. PTEN is a downstream inhibitor of the EGFR pathway and has been evaluated as a predictive factor of cetuximab efficacy in colorectal cancer. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded tumor tissue samples were collected from 226 patients with advanced or metastatic colorectal cancer that had been treated with cetuximab. Clinical information was collected retrospectively from the patients' medical records. After central evaluation, 147 cases with adequate material were eligible for further evaluation. EGFR and PTEN status was evaluated with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Data were associated with cetuximab treatment outcome. Additional analysis was performed with previously published data on PIK3CA, BRAF and KRAS mutation status and EGFR ligand amphiregulin (AREG) and epiregulin intratumoral mRNA expression levels. PIK3CA mutation status and PTEN protein expression were also analyzed as a single complex parameter, to evaluate the predictive value of PI3K/PTEN axis dysfunction as one entity. RESULTS: Analysis showed a borderline association of overall response rate (ORR) and time to progression (TTP) with EGFR protein overexpression by IHC (p = 0.059 and p = 0.057, respectively) and a positive association of EGFR gain by FISH (found in only five cases) with longer TTP (p = 0.026). No association was found between ORR or TTP and PTEN IHC or FISH status. Comparative analysis with previously published data showed that PTEN protein expression is associated with longer TTP in patients with wild-type (WT) KRAS (p = 0.036) and especially in the ones with elevated AREG levels (p = 0.046), as well as in patients with both KRAS and BRAF WT (p = 0.019). Patients with both PIK3CA WT and PTEN protein expression had significantly longer TTP (p = 0.010) versus all others, in the absence of BRAF and KRAS mutations, a finding which persisted in the KRAS WT/AREG high subgroup (p = 0.046). CONCLUSIONS: In this cetuximab-treated colorectal cancer population, EGFR gain was associated with better outcome and PTEN protein expression with longer TTP in KRAS WT, KRAS WT/AREG high and KRAS/BRAF WT subpopulations. Cetuximab efficacy is greater with intact and activated EGFR signaling, without activating mutations of KRAS/BRAF and in the presence of preserved PTEN inhibitory activity upon the PI3K/AKT pathway. These results reflect a solid biological rationale and warrant further evaluation of the predictive role of PTEN in prospective studies.

The RhoD to centrosomal duplication.

The main functional roles attributed to the centrosome, the major microtubule organizing center (MTOC) of metazoans, are related to cell locomotion, sensory perception and division. The role of vesicular trafficking in the regulation of the centrosome cycle has been largely unexplored. Recently, however, several studies have indicated the involvement of molecules and/or complexes of the trafficking routes in centrosome positioning, duplication and regulation. Functional screens have revealed communication between the outer nuclear envelope, the Golgi apparatus, the endosomal recycling compartment and centrosomes, while other studies underline the involvement of the ESCRT complex proteins in centrosome function. In this commentary, we discuss our recent study, which shows the involvement of an endosomal Rho protein, namely RhoD, in centrosome duplication and possible links between the centrosome's structural and functional integrity to vesicular trafficking.

Cytokine effects on cell survival and death of A549 lung carcinoma cells.

PURPOSE: IL-13, TNF-α and IL-1ß have various effects on lung cancer growth and death, but the signaling pathways mediating these effects have not been extensively analyzed. Therefore, the effects of IL-13, TNF-α and IL-1ß alone, and in combination with Fas, on cell viability and death as well as major signaling pathways involved in these effects were investigated in A549 lung carcinoma cells. RESULTS: Using MTT and flow cytometry, IL-13, TNF-α and IL-1ß pretreatment decreased Fas-induced cell death. These anti-cell death effects were attenuated by pretreatment with inhibitors of Nuclear factor-κB [NF-κB], Phoshatidylinositole-3 kinase [PI3-K], JNK, p38 and ERK1/2 pathways. Using Western blot, IL-13, TNF-α and IL-1ß treated cells showed time-dependent expression of p-ERK1/2, p-p38, p-JNK, p-Akt and p-IκBα proteins, decreased IκBα protein expression, no cleavage of Caspase-3 and PARP1 proteins and no notable alterations of Fas protein. IL-13 and TNF-α treated cells showed time-dependent increase of FLIPL expression. CONCLUSION: IL-13, TNF-α and IL-1ß attenuate the pro-cell death effects of Fas on A549 cells, at least partially, by pathways involving the NF-κB, PI3-K and MAP kinases, but not by alterations of Fas protein expression. The IL-13 and TNF-α cell survival effects may also be due to increased expression of FLIPL protein.

Immunohistological analysis of cell cycle and apoptosis regulators in thymus.

The combined expression patterns of cell cycle and apoptosis regulators have not been analyzed in details in human thymus to the best of our knowledge. Our objective was to provide multiparametric and combined immunohistological information regarding the expression levels and the topographical distribution of major cell cycle and apoptosis regulators in postnatal human thymus. Ki67 and cyclins A, B1, D3 and E were frequently expressed by thymocytes with higher expression in cortical than medullary thymocytes. The expression of cyclin D2 was low in thymocytes. Thymic epithelial cells (TEC) exhibited low expression of Ki67 and cyclins. Bid was frequently expressed by thymocytes, Bcl-xL by cortical thymocytes and Bcl-2 by medullary thymocytes. The expression levels of Bim and survivin in thymocytes were low. The expression levels of Bax and Mcl-1 were higher in medullary than cortical thymocytes and TEC. Bak and Bad were mainly expressed in medullary TEC and Hassall Bodies (HB). c-FLIP and Fas were frequently expressed in TEC and FasL was mainly expressed by medullary TEC and HB. Cleaved caspase-3 was expressed by scattered thymocytes at the cortex and the corticomedullary junction and very rarely at the medulla. The different expression profiles and immunotopographical distribution of cell cycle and apoptosis regulators in thymocytes and TEC indicate that their expression is tightly regulated during thymic cell differentiation and that they are differentially involved in the cell survival/death regulation of thymocytes and TEC. Furthermore, this study indicates decrease of the proliferation and caspase-dependent apoptosis of thymocytes from the cortex to the medulla.

Immunohistological characterization of thymic dendritic cells.

BACKGROUND: Dendritic cells play key roles in thymic histophysiology and histopathology. Therefore, we analyzed the immunotopographical distribution of cells expressing markers of dendritic cells and macrophages in postnatal human thymus. MATERIALS AND METHODS: The streptavidin-biotin peroxidise-labeled (LSAB) and the double-LSAB/alkaline phosphatase/anti-alkaline phosphatase (APAAP) immunohistochemical procedures were used. RESULTS: S100 protein-, Cluster of designation 1a (CD1a)-, CD207-, CD11c- and CD123-positive cells, many of them exhibiting the morphology of dendritic cells, were detected in the cortex but mainly in the medulla. These markers, except CD123, were also detected in cells of juvenile and immature Hassall bodies. CD68- and CD163-positive cells were detected in the cortex and the medulla but not in Hassall bodies. CONCLUSION: The immunohistological detection of S100-, CD1a-, CD207- and CD11c-positive dendritic cells in juvenile and immature Hassall bodies may reflect an important role of these structures in the cooperation of epithelial and dendritic cells in the process of T-cell differentiation.

Hypoxia and activated VEGF/receptor pathway in multiple myeloma.

BACKGROUND/AIM: Intensified angiogenic pathways are associated with poor prognosis and resistance of multiple myeloma (MM) cells to therapy. The links of the VEGF pathway with the hypoxia inducible factor (HIF) expression in MM are herein investigated. MATERIALS AND METHODS: The vascular density (VD) and the HIF/VEGF/VEGF-receptor expression in the bone marrows of 106 MM cases were studied using immunohistochemistry. RESULTS: HIF1alpha and HIF2alpha were expressed strongly in 33% and 13.2% of the cases, respectively. VEGFR and the phosphorylated (active) form of VEGFR2/KDR receptors were up-regulated in 42.5% and 36.8% of cases, respectively. Both HIF1alpha and HIF2alpha were significantly linked with high VD and VEGF expression. Moreover, the expression of the phosphorylated (active) form of VEGFR2/KDR was significantly linked with VEGF and HIF1alpha expression. The HIF/VEGF/VEGF-receptor pathway is up-regulated in approximately 40% of MM cases and linked with increased angiogenesis. Survival analysis in 37 evaluable patients showed a significantly worse prognosis in cases with high VD. CONCLUSION: HIFs and VEGF are up-regulated in a significant percentage of MM and are strongly related to each other. Targeting HIFs and the VEGF/receptor autocrine loop may prove of importance in the treatment of the disease.

Randomized phase II exploratory study of prophylactic amifostine in cancer patients who receive radical radiotherapy to the pelvis.

BACKGROUND: This study aimed to investigate the efficacy of prophylactic amifostine in reducing the risk of severe radiation colitis in cancer patients receiving radical radiotherapy to the pelvis. METHODS: Patients with pelvic tumours referred for radical radiotherapy who consented participation in this trial, were randomly assigned to receive daily amifostine (A) (subcutaneously, 500 mg flat dose) before radiotherapy or radiotherapy alone (R). Sigmoidoscopy and blinded biopsies were scheduled to conduct prior to initiation and following completion of radiotherapy and again 6 to 9 months later. Radiation colitis was assessed by clinical, endoscopic and histolopathological criteria. RESULTS: A total 44 patients were enrolled in this trial, the majority with rectal (20 patients) and cervical cancer (12 patients) and were assigned 23 in R arm and 21 in the A arm. In total 119 sigmoidoscopies were performed and 18 patients (18/44, 40.9%) were diagnosed with radiation colitis (15 grade 1 and 2, and 3 grade 3 and 4). Of them, 6 patients belonged to the A group (6/21, 28.6%) and 12 to the R group (12/23, 52.2%). Acute and grade IV radiation colitis was only developed in four patients (17.4%) in the R group. Amifostine side effects were mild. Amifostine treated patients were less likely to develop histologically detectable mucosal lesions, which indicate protection from acute mucosal injury. CONCLUSIONS: Amifostine given subcutaneously can lower the risk of acute severe radiation colitis in patients who receive radical radiotherapy to pelvic tumors.

High levels of topoisomerase IIalpha protein expression in diffuse large B-cell lymphoma are associated with high proliferation, germinal center immunophenotype, and response to treatment.

Gene copy number and protein expression of topoisomerase IIalpha were correlated to benefit from anthracyclines in various tumors. A retrospective series of 69 patients with DLBCL managed with CHOP chemotherapy were studied for immunohistochemical TopoIIalpha expression and numerical gene abnormalities by fluorescent in situ hybridization (FISH). The results were analyzed in relation to the expression of cell cycle proteins (Ki67, p53, HDM2, p21, p14, pRb, p16, and cyclins A, B1, D1, D2, D3, and E) and BCL6/CD10/MUM1/CD138 B-cell differentiation immunophenotype and outcome. High levels of TopoIIalpha protein were found in 91% of DLBCL cases. No evidence of TopoIIalpha gene amplification or deletion was found. The TopoIIalpha expression showed significant positive correlations with the proliferation index Ki67 (p = 0.002), cell cycle proteins pRb and cyclin D2 (p = 0.018 and p = 0.028, respectively), and the germinal center proteins bcl6 and CD10 (p = 0.010 and p < 0.0001, respectively). TopoIIalpha expression was significantly higher in germinal center B-cell like (GCB) DLBCL than in non-germinal center B-cell like (non-GCB) DLBCL (p = 0.048). TopoIIalpha protein was significantly associated with response to chemotherapy (chi(2), p = 0.024), but not with relapse-free or overall survival (p = 0.5). On multivariate analysis, only stage of disease retained independent prognostic significance (HR 0.33 for early stage, p = 0.008). Although TopoIIa gene copy number abnormalities were not found in DLBCL, high levels of protein expression are associated with GCB-cell differentiation immunophenotype, high proliferation, and response to treatment.

The role of apoptosis in the pathophysiology of Acute Respiratory Distress Syndrome (ARDS): an up-to-date cell-specific review.

ARDS pathophysiology is characterized by complex mechanisms that involve cells of inflammation, lung tissue cells, cytokines, chemokines, as well as apoptosis activators and inhibitors. There are two important theories that link apoptosis with ARDS and suggest that epithelial cell apoptosis, as well as the accumulation of neutrophils in the lung, may contribute to a cascade of events and, finally, ARDS. The activation of the Fas/FasL pathway is an important mechanism of alveolar epithelial injury in the lungs of patients with ALI. In addition, neutrophilic inflammation in the alveolar spaces is characteristic of ALI in humans and in most animal models of ALI. The enhanced phagocytosis of apoptotic neutrophils could lead to resolution of inflammation and repair during ARDS. In this review, we will focus on elucidating the role of apoptosis in the pathophysiology of ARDS and the contribution of Fas-mediated inflammation in ARDS. Furthermore, we will give evidence that TNF-alpha, IL-1beta and IL-13 attenuate the pro-cell death effects of Fas/CD95 on A549 epithelial cells, at least partially, by the NF-kB and PI3-K pathways, suggesting that induction of the expression of antiapoptotic genes protects the epithelial cells from cell death.

Concurrent development of spontaneous pyomyositis due to Staphylococcus epidermidis and Kikuchi-Fujimoto disease.

Staphylococcus epidermidis is a common cause of infections associated with prosthetic devices and immunocompromised patients. Spontaneous pyomyositis due to the above pathogen is very uncommon. Kikuchi-Fujimoto disease (KFD) is a subacute necrotizing lymphadenitis, first described in Japan. A T cell-mediated hyperimmune response to various pathogens in a genetically susceptible individual has been primarily been considered in its pathogenesis. We report a patient who developed spontaneous pyomyositis caused by S. epidermidis concurrently with KFD, and discuss the possibility of S. epidermidis infection being the stimulant of KFD.

Osteochondromas: review of the clinical, radiological and pathological features.

Osteochondroma is the most common benign bone tumor and usually occurs in the metaphyseal region of the long bones. This tumor takes the form of a cartilage-capped bony outgrowth on the surface of the bone. The vast majority (85%) of osteochondromas present as solitary, nonhereditary lesions. Approximately 15% of osteochondromas occur as multiple lesions in the context of hereditary multiple osteochondromas (HMOs), a disorder that is inherited in an autosomal dominant manner. Most lesions appear in children and adolescents as painless, slow-growing masses. However, depending on the location of the osteochondroma, significant symptoms may occur as a result of complications such as fracture, bony deformity, mechanical joint problems and vascular or neurologic compromise. Malignant transformation of osteochondromas can occur later in adulthood but rarely metastasize. The treatment of choice for osteochondroma is surgical unless the skeleton is still immature. Pathogenetic analysis showed that HMOs are caused by mutations in either of two genes: exostosis (multiple)-1 (EXT1), which is located on chromosome 8q24.11-q24.13 or exostosis (multiple)-2 (EXT2), which is located on chromosome 11p11-12. Recently, biallelic inactivation of the EXT1 locus was described in nonhereditary osteochondromas. The EXT1 and EXT2 proteins function in the biosynthesis of heparin sulfate proteoglycans (HSPGs) which are multifunctional proteins involved in several growth signaling pathways in the normal epiphyseal growth plate. Reduced EXT1 or EXT2 expression in osteochondromas is associated with disordered cellular distribution of HSPGs, resulting in defective endochondral ossification which is likely to be involved in the formation of osteochondromas. Here the clinical, radiological, pathological and pathogenetic features and the treatment modalities of osteochondroma are reviewed.

Potential value of PTEN in predicting cetuximab response in colorectal cancer: an exploratory study.

BACKGROUND: The epidermal growth factor receptor (EGFR) is over-expressed in 70-75% of colorectal adenocarcinomas (CRC). The anti-EGFR monoclonal antibody cetuximab has been approved for the treatment of metastatic CRC, however tumor response to cetuximab has not been found to be associated with EGFR over-expression by immunohistochemistry (IHC). The aim of this study was to explore EGFR and the downstream effector phosphatase and tensin homologue deleted on chromosome 10 (PTEN) as potential predictors of response to cetuximab. METHODS: CRC patients treated with cetuximab by the Hellenic Cooperative Oncology group, whose formalin-fixed paraffin-embedded tumor tissue was available, were included. Tissue was tested for EGFR and PTEN by IHC and fluorescence in situ hybridization (FISH). RESULTS: Eighty-eight patients were identified and 72 were included based on the availability of tissue blocks with adequate material for analysis on them. All patients, except one, received cetuximab in combination with chemotherapy. Median follow-up was 53 months from diagnosis and 17 months from cetuximab initiation. At the time of the analysis 53% of the patients had died. Best response was complete response in one and partial response in 23 patients. In 16 patients disease stabilized. Lack of PTEN gene amplification was associated with more responses to cetuximab and longer time to progression (p = 0.042). CONCLUSION: PTEN could be one of the molecular determinants of cetuximab response. Due to the heterogeneity of the population and the retrospective nature of the study, our results are hypothesis generating and should be approached with caution. Further prospective studies are needed to validate this finding.

Immunohistochemical expression patterns of neural and neuroendocrine markers, the neural growth factor receptors and the beta-tubulin II and IV isotypes in human thymus.

Increasing evidence suggests that neuroimmune networks play key roles in the thymic histophysiology and pathology. Prompted by this, we analyzed by immunohistochemistry the distribution of human thymic cells expressing major neural and neuroendocrine markers and neural growth factor (NGF) receptors in combination with the expression patterns of various cytokeratins. Additionally, since some beta-tubulin isotypes are preferentially expressed in neuronal cells, the immunotopographical distribution of thymic cells expressing beta-tubulin II, III and IV was analyzed. Thymic epithelial cells (TECs) expressed protein gene product 9.5 (PGP 9.5), chromogranin A (CHRA), synaptophysin (SYN), neuron-specific enolase (NSE), tyrosine hydroxylase (TH), CD56, CD57, neurofilaments (NF) (140-160 kDa), NGF receptors (TrKA and p75), beta-tubulin II and IV isotypes and cytokeratin 7, 8, 10, 13, 14, 18 and 19. PGP 9.5 was preferentially expressed in cortical TEC whereas SYN, CHRA, NSE, TH and NF 140-160 kDa were preferentially expressed in medullary TECs and Hassal corpuscles. Variable levels of expression of beta-tubulin II and IV were observed in all TEC subtypes whereas beta-tubulin III was undetectable in TECs. Subcapsular and cortical TECs display higher expression of beta-tubulin IV and lower expression of beta-tubulin II in comparison to those observed in medullary TEC and Hassal corpuscles. The diversity of the immunotopographical distibution and the expression of neural and neuroendocrine markers, the NGF receptors TrKA and p75, and the beta-tubulin II and IV isotypes in the distinct subtypes of TEC may reflect the diversity of their biological functions and/or their different stages of differentiation. The present results provide further immunohistological evidence that numerous neural and neuroendocrine factors may be required for the development and function of the human thymic microenvironment.

Variant-specific patterns and humoral regulation of HP1 proteins in human cells and tissues.

We have examined the occurrence and distribution of HP1alpha and HP1beta under in vivo, ex vivo and in vitro conditions. Consistent with a non-essential role in heterochromatin maintenance, both proteins are diminished or undetectable in several types of differentiated cells and are universally downregulated during erythropoiesis. Variant-specific patterns are observed in almost all human and mouse tissues examined. Yet, the most instructive example of HP1 plasticity is observed in the lymph nodes, where HP1alpha and HP1beta exhibit regional patterns that are exactly complementary to one another. Furthermore, whereas HP1alpha shows a dispersed sub-nuclear distribution in the majority of peripheral lymphocytes, it coalesces into large heterochromatic foci upon stimulation with various mitogens and IL-2. The effect of inductive signals on HP1alpha distribution is reproduced by coculture of immortalized T- and B-cells and can be confirmed using specific markers. These complex patterns reveal an unexpected plasticity in HP1 variant expression and strongly suggest that the sub-nuclear distribution of HP1 proteins is regulated by humoral signals and microenvironmental cues.

Serum levels, and bone marrow immunohistochemical expression of, vascular endothelial growth factor in patients with chronic myeloproliferative diseases.

Current data suggest that angiogenesis plays a significant role in the pathogenesis and progression of chronic myeloproliferative diseases (cMPDs). In the present study, we evaluated serum levels of vascular endothelial growth factor (VEGF) in 83 patients with cMPDs [myelofibrosis with myeloid metaplasia (MMM, n = 25), essential thrombocythaemia (ET, n = 40), polycythaemia vera (PV, n = 8) and chronic myeloid leukemia (CML, n = 10)] and in 27 healthy individuals. Serum VEGF levels were significantly increased in patients with cMPDs compared to healthy individuals (all p values were < or = 0.05) and were significantly correlated with bone marrow microvessel density (MVD) (p = 0.0013). In addition, the immunohistochemical expression of VEGF protein in bone marrow biopsy specimens were analyzed in 61 patients with cMPDs, (ET, n = 36 and MMM, n = 25) and in 27 healthy individuals. The cellular distribution of VEGF expression was similar in bone marrow specimens of patients and healthy individuals. VEGF protein was detected mainly in erythroid cells, whereas myeloid cells and megakaryocytes exhibited a variable expression of the protein. The percentage of bone marrow VEGF positive cells was positively correlated with serum levels of VEGF (p = 0.001). The results of the present study suggest that, VEGF is a major angiogenetic factor in patients with cMPDs and contributes to the pathogenesis of these diseases.

Expression of a molecular marker panel as a prognostic tool in gastric cancer patients treated postoperatively with docetaxel and irinotecan. A study of the Hellenic Cooperative Oncology Group.

INTRODUCTION: This study evaluated the prognostic role of vascular epidermal growth factor (VEGF), thymidylate synthase (TS), topoisomerase I (Topo-I), topoisomerase IIalpha (Topo-IIalpha) and E-cadherin (E-cadh) tumor expression, in patients with resectable gastric cancer, who were treated postoperatively with the docetaxel/irinotecan combination. PATIENTS AND METHODS: Forty-five patients with resectable gastric cancer were treated with 6 cycles of docetaxel 30 mg/m2 and irinotecan 110 m/m2 on day 1 and d8 every 21 days. All specimens were examined by using immunohistochemistry (IHC) for the expression of VEGF, TS, Topo-I, Topo-IIalpha and E-cadh. RESULTS: Positivity for TS was significantly correlated with age and for VEGF with diffuse histological type and good PS. No significant correlation was observed among Topo-I, Topo-IIalpha and E-cadh positivity with any of the clinicopathological parameters studied. Median overall survival (OS) was 31.7, and disease-free survival (DFS) 26 months, respectively. None of the above-investigated molecular markers were significantly associated with OS and DFS. Finally, according to the univariate analysis for survival, only advanced stages (III, IV) of the disease implied risk of death, mainly due to lymph node involvement and, to a lesser extent, tumor size. None of the studied molecular markers were found to be independent prognostic markers. CONCLUSION: These results should be interpreted very cautiously, due to the limited number of patients studied, as well as the limitations of the IHC technique.

Alterations of the p53, Rb and p27 tumor suppressor pathways in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphomas (DLBCL) display defects in cell cycle and apoptosis regulation. Therefore, the immunohistochemical expression patterns of the proteins p14, p21, Hdm2 and cyclin D2 were analyzed in relation to the previously reported expression of other major cell cycle proteins (p53, Rb, p16, p27, Ki-67 and cyclins A, B1, D2, D3 and E), apoptosis-associated proteins (bcl2, bcl-xl, bax, bak, bad and bid) and the B-cell differentiation immunophenotypes. Expression of the proteins p14, p21, Hdm2 and cyclin D2 was observed in 62/71 (87%), 22/76 (29%), 35/74 (47%) and 11/77 (14%) cases, respectively. Immunohistochemical alterations of the p53 (p53-Hdm2-p21-p14), Rb (Rb-p16-cyclin D [D2 or D3]) and p27 (p27-cyclin E) pathways were found in 56/77 (73%), 53/79 (67%) and 54/79 (68%) cases, respectively. Concomitant alterations of the p53-Rb, p53-p27 and Rb-p27 pathways were found in 40/77 (52%), 38/77 (50%) and 36/79 (46%) cases, respectively. Three concomitant alterations of the p53-Rb-p27 pathways were found in 28/79 (35%) cases. The main findings of the present study were the following: alterations of the p27 pathway were associated with higher expression of Ki-67 (p = 0.023); concomitant alterations of the p53Rb pathways and the p53-p27 pathways were associated with higher expression of cyclin A (p = 0.015 and p = 0.021, respectively) and concomitant alterations of the p53, Rb and p27 pathways were associated with higher expression of cyclin A (p = 0.013). Since cyclin A supports DNA replication, centrosome duplication and mitosis, these findings indicate that concomitant alterations of the p53, Rb and p27 pathways in DLBCL may have cooperative effects resulting in increased neoplastic cell proliferation. This might explain, at least partially, the association between concurrent aberrations of the p53, Rb and p27 pathways and aggressive clinical behavior in DLBCL.

A tree-based decision rule for identifying profile groups of cases without predefined classes: application in diffuse large B-cell lymphomas.

In this paper, we examined the utility of a forward growing classification tree as a supplement to cluster analysis for deriving a decision rule for the identification of profile groups when the cases do not belong to predefined classes. The technique was applied for the identification of low and high proliferation profile groups of diffuse large B-cell lymphomas according to the immunohistochemical expression levels of proliferation proteins. In a forward growing classification tree method, the size of the tree is controlled by the improvement (threshold value) in the apparent misclassification rate after each split. The classes used in the tree were defined using k-means clustering. The decision rule consisted of the splitting points of the split variables used. The methodology was applied to the histology data from 79 cases of diffuse large B-cell lymphomas. Ten classes of individual cases were derived from k-means clustering. Then, a classification tree with a threshold of 2% was used to derive the decision rule. Branches at the left side of the tree consisted of individuals with a low proliferation profile and branches at the right side of the tree consisted of cases with a high proliferation profile. The classification tree, as a supplement method, not only identified but also provided decision rules for identifying profile groups. Finally, it also allowed for exploration of the data structure.