Malignant Cell Clone Research Articles

Stromal cell-derived factor-1 chemokine gene variant in blood donors and chronic myelogenous leukemia patients.

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disorder that originates from a pluripotent stem cell expressing the bcr-abl oncogene. It is characterized by an abnormal release of the expanded, malignant stem cell clone from the bone marrow into the circulation. The stromal cell derived factor-1 (SDF-1) gene contains a common polymorphism, termed SDF1-3'A, in an evolutionarily conserved segment of the 3' untranslated region (UTR). In this work the SDF-1 genotypes of 25 patients (9-82 years old) who had been clinically and hematologically diagnosed with CML were compared with those of 60 healthy donors. In addition, the nature of bcr-abl hybrid mRNA and the association between demographic and hematological parameters were analyzed in cells from 12 CML patients (five women and seven men). All patients underwent blood collection during the chronic phase of disease after they received chemotherapy. b3a2 mRNA was detected in samples from eight of the CML patients and b2a2 mRNA was observed in four cases. An association between basophils and hemoglobin parameters was observed in that hemoglobin levels were higher in b2a2-expressing patients, and mean basophil levels were higher in patients expressing b3a2. Four of the CML patients (16%) were homozygous for 3'A allele. Of the patients who showed the presence of bcr-abl transcripts (N = 12), three presented the wt/wt genotype and nine were SDF1-3'A carriers. Three of the latter were homozygous for this mutation. It is possible that the bcr-abl fusion gene and the SDF1 genotype for 3'A allele have important implications for the pathogenesis of CML.

A sexually transmitted parasitic cancer

Cancer is not a contagious disease but two exceptions have come to light, in the dog and in the marsupial Tasmanian devil. Canine transmissible venereal tumor (CTVT) is sexually transmitted between dogs and was the first tumor to be experimentally transplanted in 1876. Using forensic DNA markers we have shown [1] that a single malignant clone of CTVT cells has colonized dogs worldwide. Thus the tumor has evolved into an independent parasite, which has long outlived its original host, possibly a wolf. CTVT represents the oldest known malignant cell in continuous propagation, and challenges the concept of increasing genome instability in cancer progression. As an allograft, CTVT defies natural transplantation barriers. Can human tumor cells similarly be transmitted from one person to another?

Comprehensive analysis of 130 multicentric intraepithelial female lower genital tract lesions by HPV typing and p16 expression profile

HPV associated cervical transformation is characterized by well-defined steps, including persistent HPV infection and deregulated oncogene expression. Recent studies have suggested that a number of lower genital tract lesions are clonally related to cervical lesions. In the current study, HPV infections and oncogene expression were assessed in a large series of patients with multicentric lower genital tract disease to analyze the transformation steps in extracervical disease. One hundred and thirty biopsies of 52 women treated for multicentric synchronous or metachronous lower genital tract intraepithelial neoplasias were collected. Up to seven multicentric specimens taken from one patient were studied with a maximum follow up of 20 years. HPV typing and p16(ink4a) immunostaining was performed. HPV DNA was present in 121 of 130 specimens (93%). HPV16 was frequently found in VIN, VaIN and AIN (73, 60 and 77%, respectively), whereas only 37% of CIN were HPV16 positive. Infections with identical HPV types in multicentric lesions were diagnosed in 46% of the HPV positive patients. p16INK4a expression was negative in the nine HPV negative lesions whereas about 90% of the high grade lesions showed diffuse p16 staining. Our findings indicate that multicentric lower genital tract disease evolves through different pathways. Some cases were related to a high susceptibility towards HPV infections, while others exhibited features of clonal propagation of transformed cervical cell clones. The clinical management of the latter group is particularly challenging, because malignant cell clones can persist over a long time course.

Modeling BCR-ABL Transcript Dynamics in CML Patients under Imatinib Treatment.

Treatment of chronic myeloid leukemia (CML) with the tyrosine kinase inhibitor imatinib represents a successful application of a molecularly targeted therapy. A rapid hematologic and cytogenetic response can be induced for the majority of patients even in advanced disease. However, the time course of disappearance of leukemia cells, characterized by the expression of the BCR-ABL fusion protein, varies between patients, and a complete eradication of the malignant cells is a rare event. The reasons for the heterogeneous response and the persistence of the malignant clone in many patients are currently not known.We propose a mathematical model which consistently explains short and long-term dynamics of BCR-ABL transcript levels in populations of CML patients under imatinib monotherapy. The model is based on the concept that normal and malignant cell clones compete for growth environments in which they behave slightly differently with regard to homing and cell cycle activation/deactivation. This concept has been successfully applied for understanding time-dependent chimerism in mice [Roeder et al.: Blood 105(2):609]. Applying the model to data sets from two independent cohorts of imatinib treated CML patients, we demonstrate the potential of our model to quantitatively describe the typical biphasic decline in BCR-ABL transcript levels during the first year of treatment. Besides the median transcript dynamics in the patient population the model is able to represent the heterogeneity in individual transcript time courses. Qualitative differences in the imatinib response are explained by small quantitative differences in the drug effects regarding proliferation inhibition and/or induction of apoptosis for BCR-ABL positive cells. As demonstrated by comparison with five years follow-up data of 69 unselected newly diagnosed CML patients recruited into the IRIS trial in Germany [Mueller et al.: Leukemia 17(12):2392] the model also correctly describes long-term BCR-ABL dynamics. The observed median BCR-ABL transcript levels, including the vanishing decline after year four of treatment, can quantitatively be explained by a decreasing treatment efficiency in a subset of patients, potentially caused by imatinib-resistant clones. Sensitivity analyses show that moderate functional differences of the resistance mutations can lead to remarkable differences in long-term treatment efficiency. On the other hand, in patients not developing resistance mutations our model predicts the general chance of an eradication of the malignant clone in the long run. This is supported by data in a patient subgroup showing a continued decline of BCR-ABL transcript levels over five years of treatment.Beyond the consistent description of the clinically observed BCR-ABL dynamics we provide testable predictions for effects of different combination treatments. Based on the explanation of CML as a clonal competition of malignant and normal hematopoietic stem cells, our model particularly predicts that the therapeutic benefit of imatinib can be augmented by a combination with proliferation stimulating treatment strategies. In addition the model permits to describe the heterogeneity of the effect of resistance mutations with respect to specific treatment strategies. In summary, our model describes CML dynamics under imatinib therapy with potential implications for the design of future treatment strategies.

The dawn of prophylactic vaccines against human papillomaviruses and cervical cancer

The dawn of prophylactic vaccines against human papillomaviruses and cervical cancer

Apoptosis in Physiological and Pathological Skin: Implications for Therapy

Apoptosis is an inducible suicide program that occurs in all phases of multicellular as well as in protozoa life and gains more and more importance in all medical disciplines. It is required for normal ontogenesis, organ and tissue remodeling, function of the immune system, prevention of inappropriate cellular proliferation and of survival of inappropriate mutations. Thereby apoptosis represents the key event which guarantees differentiation and maintenance of homeostasis. Terminal differentiation seems to be a special form of apoptosis. Dysregulated apoptosis is associated with various pathological conditions, including inflammation, and cancer. Acanthosis, the hallmark of psoriatic skin, is an example for diminished epidermal apoptosis. Defects in termination of inflammatory reactions occur in atopic dermatitis. Lupus erythematosus may arise due to disturbed apoptosis on several check points of the apoptosis cascade. Experimental evidence suggests a role for Bcl-2 and CD95L in the inhibition of programmed cell death in UV-induced skin cancer or malignant melanoma cells. Thus, it leads to survival of malignant cell clones. The slow growth of basal cell carcinomas is due to an increased apoptosis to mitosis ratio. Spontaneous regression of tumors is associated with increased apoptotic rates. Malignant melanoma cells characteristically show different anti-apoptotic strategies which underscore its aggressive behavior and its refractory towards classic therapeutic regimens. Additionally, induction of apoptosis in tumor infiltrating immune cells seems to be a strategy by which the tumor escapes from an immunological attack (tumor counter-attack). Since apoptosis is either absent or altered under pathological conditions therapeutic procedures should correct this. Established therapies like dithranol, vitanin-D3 analogs, low-dose methotrexate, induce apoptosis. Future treatment regimens like vaccine and gene therapy are designed to selectively induce apoptosis. Therefore, pharmacological agents and therapeutic strategies interfering with disrupted apoptosis regulation could improve the therapeutic arsenal in the future.

Adding Chloroquine to Conventional Treatment for Glioblastoma Multiforme

Malignant cell clones resistant to chemotherapy and radiotherapy frequently lead to treatment failure in patients with glioblastoma multiforme. Preliminary studies suggest that adding chloroquine to conventional therapy may improve treatment outcomes. To examine the effect of adding chloroquine to conventional therapy for glioblastoma multiforme. Randomized, double-blind, placebo-controlled trial. National Institute of Neurology and Neurosurgery of Mexico. 30 patients with surgically confirmed glioblastoma confined to 1 cerebral hemisphere, with a Karnofsky performance score greater than 70, no comorbid disease, and age younger than 60 years. Oral chloroquine at 150 mg/d for 12 months beginning on postoperative day 5 or placebo. All patients received conventional chemotherapy and radiotherapy. Primary outcome was survival after surgery; surviving patients were followed up to October 2005. Periodic evaluation using the Karnofsky scale and imaging studies, as well as hematologic tests and ophthalmologic examinations, was done in all patients. Median survival after surgery was 24 months for chloroquine-treated patients and 11 months for controls. At the end of the observation period, 6 patients treated with chloroquine had survived 59, 45, 30, 27, 27, and 20 months, respectively; 3 patients from the control group had survived 32, 25, and 22 months, respectively. Although not statistically significantly different, the rate of death with time was approximately half as large in patients receiving chloroquine as in patients receiving placebo (hazard ratio, 0.52 [95% CI, 0.21 to 1.26]; P = 0.139). The observed advantage of chloroquine may be due to chance; differences in pretreatment characteristics and conventional treatment regimens could not be adjusted for because of the small sample size. Chloroquine may improve mid-term survival when given in addition to conventional therapy for glioblastoma multiforme. These results suggest that larger, more definitive studies of chloroquine as adjuvant therapy for glioblastoma are warranted.

Low Dose Thalidomide for the Treatment of Multiple Myeloma.

In the last two decades, we have seen a radical change in the therapy and prognosis of Multiple Myeloma (MM). Recent research on the role of the bone marrow microenvironment as integrant part of the biology of MM and the possible therapeutic interference at this level are leading to control and regression of the malignant plasma cell clone with a recognized clinical impact. Thalidomide, an old drug thought to interfere on the bone marrow microenvironment, is active not only for the treatment of refractory patients but also, as was recently shown, for induction and/or remission maintenance therapy. Most of the side effects (somnolence, constipation, fatigue, tremor, bradycardia, edema, and neuropathy) can be managed by reducing the dose and most patients need a dose reduction. The appropriate dose of thalidomide in myeloma is unknown. We treated thirty-five patients with MM with low-dose thalidomide (200mg or less). Twenty four patients were on maintenance therapy (13 after bone marrow transplantation, and 11 after chemotherapy with VCAP/VMCP); 5 patients were treated after relapse, 4 with refractory disease and 2 for remission induction as a first line therapy. Patients were treated at the Hematology and Bone Marrow Transplantation Service of the Hospital de Clínicas de Porto Alegre, RS, Brazil, from march/2001 to December/2003. The response as measured by hemoglobin level, immunoglobulin (M component) or urinary light chain concentration and bone marrow examination was evaluated before, 3, 6, and 12 months after the beginning of thalidomide.Results: All patients are alive and well. Fifty one percent are on 100mg schedule; of the patients on maintenance therapy 90% are on complete remission or on a sustained plateau. A Wilcoxon ranks test for the immunoglobulin level before and after 6 months for the entire group showed p=0.001 (25–75%).Conclusion: Low dose thalidomide is tolerable and effective.

Abnormal PI 3-Kinase Activity Due to Increased Lyn with Decreased PTEN and Absent SHIP in Bone Marrow Cells from Patients with Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) result from a malignant stem cell clone characterized by ineffective hematopoiesis, manifested as peripheral cytopenia with a cellular bone marrow. A leading hypothesis is that MDS results from a breakdown in the control of myeloid cell proliferation and apoptosis. Through its generation of 3′-phosphoinositides and subsequent activation of effectors such as Akt, phosphatidylinositol 3-kinase (PI 3-kinase) drives cell proliferation, apoptosis, differentiation, and motility. We show here that PI 3-Kinase is profoundly deregulated in high-risk MDS. Bone marrow cells from high-risk MDS patients displayed a 3–30 fold increase in constitutive activation of the Src kinase Lyn. Constitutive serine/threonine phosphorylation of Akt, a surrogate of PI-3 kinase activity, was seen in all specimens. Protein levels of PTEN, which dephosphorylates the D3-position phosphate of the inositol ring, were variably decreased. Since PTEN is frequently silenced by hypermethylation, we treated U937, THP-1, and Mo7e cells with 5-azacytidine. However, real-time PCR showed no increase in PTEN transcripts in these cell lines. More significantly, protein expression of SHIP-1, which dephosphorylates the D5-position phosphate of the inositol ring, was markedly decreased or absent in bone marrow cells from MDS patients, whereas they were present in AML or ALL blasts. Real-time PCR showed SHIP-1 transcripts in MDS cells to be 50% of normal CD34+ stem cells. Although 5-azacytidine treatment resulted in an increase in SHIP-1 transcripts, as measured by quantitative PCR, protein levels did not increase. Because SHIP-1 contains three PEST-rich regions, we hypothesized that the low or absent level of expression of SHIP-1 may due to protein degradation. Neither the cryopreservation of cells nor lysis buffer could explain the absence of SHIP-1 protein. Instead, when U937 and HL60 leukemic cell lines were treated with the proteastome inhibitor lactacystin (1–5 uM), protein levels of SHIP-1 increased. These results suggest that constitutive activation of Akt is likely due to decreased PTEN and absent SHIP-1 protein levels in primary MDS cells. Consistent with these findings, mice deficient in PTEN and SHIP-1 suffer from anemia, thrombocytopenia, leukocytosis and impaired function of myeloid progenitors (BLOOD 103:4503, 2004). These results also suggest that a combination of a Src kinase inhibitor and a proteasome inhibitor may be a therapeutic approach for the treatment of high-risk MDS.

Identification of MDS-Specific and Methylation Associated Downregulation of Survivine and WT1 in Highly Purified Hematopoietic Progenitor Cells during In Vitro Lineage Specific Differentiation.

The molecular pathogenesis of myelodysplastic syndromes (MDS) is poorly understood. To detect epigenetic alterations of key regulatory genes during lineage-specific hematopoietic differentiation, CD34+ cells from healthy donors (n=7) and MDS patients with low risk (LR; RA/n=6, RARS/n=3) and high risk (HR; RAEB/n=4, RAEB-T/n=2) MDS according to IPSS were in vitro differentiated with EPO, TPO or GCSF and harvested at days 0, 4, 7 and 11. For each lineage at each time point, promotor methylation analysis of genes involved in cell cycle control (p14, p15, p16, CHK2), DNA-repair (hMLH1), apoptosis (p73, survivine, DAPK) and differentiation (RARb, WT1) was performed by methylation specific PCR (MSP) of bisulfite converted genomic DNA. Corresponding gene expression was analyzed by oligonucleotide microarrays. Remarkably in CD34+/d0 cells p16 was methylated in 0% controls in contrast to 22% in MDS LR- and 50% in MDS HR-patients. Likewise in CD34+/d0 cells WT1 methylation was found in 0% controls but in 62% MDS LR- and 33% MDS HR-patients. During differentiation (d4, d7, d11) survivine promoter methylation was found in all 3 hematopoietic lineages in MDS only (LR/EPO: 22%, LR/TPO: 11%, LR/GCSF: 55%; HR/EPO: 67%, HR/TPO: 33%, HR/GCSF: 67%). WT1 promotor methylation during differentiation was almost absent in controls (EPO: 0%, TPO: 0%, GCSF: 29%) in contrast to MDS (LR/EPO: 22%, LR/TPO: 11%, LR/GCSF: 55%; HR/EPO: 17%, HR/TPO: 17%, HR/GCSF: 67%). CHK2 methylation during differentiation was not found in controls but in MDS patients (LR/EPO: 44%, LR/TPO: 22%, LR/GCSF: 22%; HR/EPO: 33%, HR/TPO: 33%, HR/GCSF: 50%). Interestlingly, mRNA expression of survivine was significantly reduced in methylated samples during erythropoiesis (p=0,04). Unexpectedly p73 RNA-expression was lower during granulopoiesis (p=0,052) in unmethylated samples. Similarly p16 mRNA showed significantly reduced expression during erythropoiesis (p=0,034) and on day 10 (p=0,064) and day 14 (p=0,035) during lineage specific differentiation in general in unmethylated specimen. Furthermore in low risk MDS WT1 RNA-expression was significantly reduced for methylated samples (p=0,01). P16 hypermethylation of CD34+/d0 cells shows a trend to correlate with MDS IPSS, but higher p16 mRNA expression values were found in methylated cases. Similarly a trend for IPSS risk correlation is shown for survivine promotor methylation during lineage specific differentiation, making it a potential marker of MDS progression, which leads to reduced mRNA expression particularly in erythropoiesis. For WT1 and CHK2 methylation patterns specific for MDS were identified, corresponding to reduced expression in WT1 promotor methylated LR-MDS. The detailed exploration of epigenetic alterations of these genes during early hematopoesis in MDS may give new insights into the pathophysiology of MDS. In particular this study identified survivine and WT1 as genes with lower methylation associated expression in MDS hematopoietic precursor cells which may ultimately contribute to the clonal expansion of the malignant cell clone.

Studies of the Site and Distribution of CD34+ Cells in Idiopathic Myelofibrosis

The frequency and distribution of CD34+ cells in the bone marrow (BM) of patients with idiopathic myelofibrosis (IM) were determined using an immunohistochemical technique. The percentage and absolute number of circulating CD34+ cells were enumerated. Patients with IM exhibited a continuum of number of BM CD34+ cells ranging from 1 to 85 per 5 mm2. The frequency of BM CD34+ cells was inversely related to the number of circulating CD34+ cells. The BM biopsy specimens obtained from 4 patients with IM who underwent allogeneic stem cell transplantation were examined sequentially. Quantitative measurement revealed that the reticulin fiber volume was progressively reduced after allogeneic stem cell transplantation. All 4 patients had normocellular marrow with normal numbers of BM CD34+ cells after transplantation. These findings suggest that the BM fibrosis and abnormal hematopoietic stem cell distribution in patients with IM is a consequence of the progeny of a malignant hematopoietic stem cell clone.

Studies of the Site and Distribution of CD34+ Cells in Idiopathic Myelofibrosis

The frequency and distribution of CD34+ cells in the bone marrow (BM) of patients with idiopathic myelofibrosis (IM) were determined using an immunohistochemical technique. The percentage and absolute number of circulating CD34+ cells were enumerated. Patients with IM exhibited a continuum of number of BM CD34+ cells ranging from 1 to 85 per 5 mm(2). The frequency of BM CD34+ cells was inversely related to the number of circulating CD34+ cells. The BM biopsy specimens obtained from 4 patients with IM who underwent allogeneic stem cell transplantation were examined sequentially. Quantitative measurement revealed that the reticulin fiber volume was progressively reduced after allogeneic stem cell transplantation. All 4 patients had normocellular marrow with normal numbers of BM CD34+ cells after transplantation. These findings suggest that the BM fibrosis and abnormal hematopoietic stem cell distribution in patients with IM is a consequence of the progeny of a malignant hematopoietic stem cell clone.

Neoplastic growth: The consequence of evolutionary malignant resistance to chronic damage for survival of cells (review of a new theory of the origin of cancer)

In the present review, a new theory that the mechanisms of general evolutionary persistent resistance to damaging factors are closely related to the development of tumour cells is introduced. Evolutionary resistance and its variability have an immense power to drive and control the process of carcinogenesis and the success of microbial and antitumour chemotherapy. First, this phenomenon of adaptation is characteristic of microbial cells whose resistance to antibiotics and other chemotherapeutic drugs is manifested through ATP-dependent transmembrane transporters. The structure and function of some multidrug transporters of resistance are conserved from microorganisms to mammals. When somatic cells are exposed to carcinogens and develop into tumour cells, they also acquire resistance to the toxic effects of carcinogens through these same transmembrane transporters (P-glycoprotein, glutathione S-transferases and other products of evolutionary resistance-related genes arisen for detoxification and exportation of cytotoxic xenobiotics and drugs). Cancerous cells acquire a persistent evolutionary resistance to chemotherapy drugs or irradiation through the same ATP-dependent transporters encountered in prokaryotic and eukaryotic cells. The mechanism of acquired resistance of cells to damaging factors, which becomes manifested during tumorigenic process formation, is a general biological law of primary significance in carcinogenesis. This resistance can be called malignant as, once formed, it does not disappear, as does also a clone of malignant cells. In tumorous cells, the mutagenic processes, morphological and functional modifications are a mechanism of secondary significance in carcinogenesis, contributing to formation of damage-resistant cells. This mechanism characterizes the processes of simplification arising in damage-resistant cells. Such cells acquire parasitic features. To survive under unfavourable conditions, they get adapted as if returning down the evolutionary stairs back to a more primitive stage of atavistic regression, which is characteristic of primitive forms of existence. Therefore they cease obeying the growth-regulating mechanisms in the organism and acquire the potential of unlimited division and accelerated growth (metastases) as do unicellular organisms or their forms resistant to damaging factors in the environment and in the host organism. Thus, cancer is a natural self-protective response of the damaged cells to the biological, physical and chemical damage and oxidative stress. This response has been developed in the process of evolution under the impact of the general biological Darwinian law of nature--to survive through variability and adaptation to the changed environmental conditions. Thus, malignization is the consequence of an evolutionary variety of the general biological resistance of cells to damage and stress in order to survive.

Genome analysis and gene expression profiling of neuroblastoma and ganglioneuroblastoma reveal differences between neuroblastic and Schwannian stromal cells

Neuroblastic tumours are a group of paediatric cancers with marked morphological heterogeneity. Neuroblastoma (Schwannian stroma-poor) (NB-SP) is composed of undifferentiated neuroblasts. Ganglioneuroblastoma intermixed (Schwannian stroma-rich) (GNBi-SR) is predominantly composed of Schwannian stromal (SS) and neuroblastic (Nb) cells. There are contrasting reports suggesting that SS cells are non-neoplastic. In the present study, laser capture microdissection (LCM) was employed to isolate SS and Nb cells. Chromosome 1p36 deletion and MYCN gene amplification were found to be associated in two out of seven NB-SPs, whereas no abnormalities were observed in five GNBi-SRs. In some cases, loss of heterozygosity (LOH) at 1p36 loci was detected in Nb cells but not in the bulk tumour by LCM; furthermore, LOH was also identified in both SS and tumour tissue of a GNBi-SR. DNA gain and loss studied by comparative genomic hybridization were observed at several chromosome regions in NB-SP but in few regions of GNBi-SR. Finally, gene expression profiles studied using an oligo-microarray technique displayed two distinct signatures: in the first, 32 genes were expressed in NB-SP and in the second, 14 genes were expressed in GNBi-SR. The results show that NB-SP is composed of different morphologically indistinguishable malignant cell clones harbouring cryptic mutations that are detectable only after LCM. The degree of DNA imbalance is higher in NB-SP than in GNBi-SR. However, when the analysis of chromosome 1p36 is performed at the level of microdissection, LOH is also observed in SS cells. These data provide supportive evidence that SS cells have a less aggressive phenotype and play a role in tumour maturation.

Mesenchymal Stem Cells of Patients with Chronic Myeloid Leukemia Are Philadelphia Negative.

In the last years, focus of regenerational studies has pointed on mesenchymal stem cells (MSC) and their ability to differentiate into several mesenchymal tissues. MSC have been shown to play a pivotal role in the microenvironment of bone marrow cells and in the modulation of immune response as they can suppress lymphocytic proliferation in vitro. Moreover, some animal studies have suggested they could favor the proliferation of malignant cell clones in solid tumor models. Their role in hematological malignancies, however, remains to be further elucidated and little is known about the influence of MSC in the development and maintenance of the malignant clone in chronic myeloid leukemia (CML). This disease is characterized by the presence of the Philadelphia (Ph) chromosome, a fusion product generated by the reciprocal translocation between chromosomes 9 and 22. Previous reports showed that hepatocytes precursors, found in the liver of CML patients carry the Ph translocation. Our intent was to elucidate whether MSC isolated from patients with CML in different stages of the disease originate from the malignant clone. To this purpose bone marrow aspirates of 11 patients with CML were obtained after informed consent. Five patients were analyzed at diagnosis, two after allogenic stem cell transplantation, three on treatment with the tyrosine kinase inhibitor imatinib and one on treatment with interferon alpha in combination with hydroxyurea. MSC were then generated as previously described. Briefly, cells were isolated by density gradient methods, resuspended in RPMI1640 medium containing 10% fetal bovine serum and plated in culture flasks to adhere. After 4–5 weeks of culture cells were collected and characterized by the expression of several surface markers in a fluorescence activated cell sorter (FACS). The presence of the Ph chromosome was assessed by both fluorescence in situ hybridization (FISH) and polymerase chain reaction (nested PCR). Moreover whole bone marrow was analyzed and results compared with those obtained in the MSC population. MSC showed a typical morphological pattern, growing to confluence after a few weeks of culture and appearing as an adherent, spindle shaped cell layer. In FACS they stained positive for SH2 and SH3 and did not express CD34, CD45 and CD14. MSC were then analyzed by FISH using probes for BCR-ABL. We could not detect the Ph translocation in any of the analyzed patients, though it was present at variuos levels in the remnant bone marrow cells. Results did not change, if expression of BCR-ABL was measured by high sensitivity RT-PCR. Our results showh that MSC of patients with CML are Philadelphia negative irrespective of the stage of disease and the treatment given, suggesting that these cells are not involved in the development of the malignancy. However, their interactions with leukemic cells as well as their role in the immune response against the tumor remains to be further characterized.

Can CD3 +/HLA-DR + activated T cells predict the prognosis of non-Hodgkin's lymphoma patients?

The immune system has several mechanisms to fight against developing malignant cell clones in the host, one of them is the activated T-cell response. Both CD4 + helper and CD8 + cytotoxic T-cells bear HLA-DR molecules as important surface activation markers. Our aim was to determine, how the ratio of activated T cells change in the peripheral blood of non-Hodgkin's lymphoma (NHL) patients during the periods of polychemotherapy. Using the methods of immunofluorescence staining and flow cytometry, we found the ratio of CD3 +/HLA-DR + cells significantly higher in NHL patients before treatment compared to healthy controls (10.63% versus 2.97%, P < 0.001). During the period of polychemotherapy, this ratio began to increase significantly (10.63% versus 16.94%, P = 0.006). After treatment, the ratio of activated T cells decreased, however, we detected significantly higher rate of CD3 +/HLA-DR + lymphocytes in patients who relapsed within 1 year than in those who stayed in remission (9.55% versus 20.62%, P < 0.001). We suppose that investigation of CD3 +/HLA-DR + activated T cells might be a promising method to determine the prognostics of lymphoma patients.

CD158k/KIR3DL2 Is a New Phenotypic Marker of Sezary Cells: Relevance for the Diagnosis and Follow-Up of Sezary Syndrome

CD158k molecules belong to the family of killer cell immunoglobulin-like receptors (KIR) that are expressed on a minor population of circulating NK and CD8+ T lymphocytes. Here, we report a strong positive correlation between the percentage of CD158k+ blood lymphocytes analyzed by flow cytometry and the percentage of atypical circulating cells (Sezary cells) determined by cytomorphology in a large group of patients with Sezary syndrome. Moreover, we show that circulating CD4+CD158k+ lymphocytes correspond to the malignant clonal cell population. Our findings suggest that the CD158k marker could be a useful tool for the evaluation of the circulating tumoral burden and the follow-up of patients with Sezary syndrome.

Growth of malignant oral epithelial stem cells after seeding into organotypical cultures of normal mucosa.

Oral squamous cell carcinoma (OSCC) is associated both with the local expansion of clones of malignant cells and with their further migration to regional and distant sites. The interactions that occur between normal and malignant cells during these events are not well modelled by standard culture conditions, but organotypical cultures, in which epithelial cells are grown on a matrix containing fibroblasts, provide a suitable environment for such investigations. Cells from five cell lines, each derived from OSCC and marked by retroviral transduction with alkaline phosphatase, were incorporated as small subpopulations (0.1-5%) in uniformly differentiating organotypical cultures constructed from normal oral mucosal cells. The patterns of growth of the malignant cells within the normal epithelium were examined for 3 weeks. There was variation between the different cell lines in their rates and patterns of growth, but all cell lines produced clusters of malignant cells that had expanded within 3 weeks to replace the normal epithelium. The appearance and spacing of these clusters suggested that each was derived from a single progenitor cell. The number of malignant cells initially present within a given area of organotypical epithelium was much greater than the number of expanding cell clusters subsequently formed. Cluster-forming cells thus represented only a subpopulation of the tumour cells. The organotypical model allows examination of interactions occurring between cells derived from OSCC and normal epithelia. The three-dimensional nature of organotypical cultures, together with their more normal patterns of differentiation, provides an environment that more closely mimics the in vivo environment in which tumours develop. The finding that only a subpopulation of tumour cells forms expanding tumour colonies suggests a range of growth potentials within a tumour population and may provide preliminary evidence for some form of stem and amplifying cell pattern.

Interaction of cutaneous lymphoma cells with reactive T cells and dendritic cells: implications for dendritic cell-based immunotherapy

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of skin neoplasms that originate from T lymphocytes. An anti-CTCL T-cell immunity has been described but seems to be inefficient to clear CTCL cells. It is not known whether cutaneous dendritic cells (DCs) perpetuate the proliferation of the malignant CTCL cell clone or play a role in the control of this usually slowly progressing disease. To characterize CTCL cell properties in the control of anti-CTCL T cells and to pave the way for a DC-based immunotherapy for CTCL. We studied the interaction of a CTCL cell line with DCs and with allogeneic T cells. We found an antigen non-specific capacity of viable but not apoptotic CTCL cells to hamper CD4+ and CD8+ T-cell proliferation in a dose-dependent manner, indicating a suppressive potential of CTCL cells. Both viable and apoptotic CTCL cells were phagocytosed by immature DCs but only apoptotic CTCL cells induced an upregulation of DC maturation markers to a degree which enabled classification of these DCs as semimature. CTCL cells did not respond with proliferation when encountering allogeneic, mature DCs either loaded with CTCL cell material or unloaded, indicating a role for DCs in the induction of anti-CTCL T-cell immunity rather than in perturbation of clonal proliferation. For the loading of DCs with CTCL material lysate seems to be optimal as apoptotic cells were not phagocytosed extensively and necrotic CTCL material induced a partial cellular toxicity in DCs. DCs loaded with CTCL material were cryopreservable without significant loss of DC viability, surface marker expression or allostimulatory activity. Together, these data argue in favour for a DC-based immunotherapy for CTCL patients and provide an experimental protocol for preparing CTCL cell-loaded DCs.

Recent advances in understanding the pathogenesis of anemia in multiple myeloma.

Anemia is a prominent feature of multiple myeloma (MM) and is commonly associated with clinical progression of MM. In addition to being affected by a number of pathogenetic events, including imbalance of the cytokine network, inappropriate erythropoietin (EPO) levels, blood loss, and hemolysis, the erythroid matrix is chronically deteriorated by the malignant plasma cell clone that activates a cytotoxic mechanism directed at the erythroid progenitors. In particular, malignant plasma cells express very high levels of apoptogenic receptors, including both Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand, which trigger apoptosis of immature erythroblasts by stimulating specific death receptors, namely Fas and the complex DR4/DR5. Erythroid cells also weakly express the transcription factor GATA-1, which drives erythroblast maturation by inhibiting apoptosis through antiapoptotic molecules such as EPO and Bcl-xL. This newly discovered pathogenetic mechanism of anemia in MM is based on persistent erythroblast cytotoxicity within the bone marrow that leads to progressive destruction of the erythroid matrix.