Tumor Cell Load Research Articles

A microfluidic system for investigation of extravascular transport and cellular uptake of drugs in tumors

Three-dimensional (3D) tumor models have been established in various microfluidic systems for drug delivery and resistance studies in vitro. However, one of the main drawbacks of these models is non-uniform distribution of cells, leaving regions with very low cell density within the 3D structures. As a result, molecular diffusion in the cell compartments is faster than that observed in solid tumors. To solve this problem, we developed a new technique for preparation of 3D tumor models in vitro. It was based on a microfluidic device containing three parallel channels separated by narrowly spaced posts. Tumor cells were loaded into the central channel at high density. To test the system, B16.F10 melanoma cells were perfusion-cultured overnight and the resulting 3D structure was characterized in terms of viability, density, and morphology of cells as well as transport properties of small fluorescent molecules. Immediately upon loading of tumor cells, the cell density was comparable to those observed in B16.F10 tumor tissues in vivo; and the viability of tumor cells was maintained through the overnight culture. The tumor model displayed low extracellular space and high resistance to diffusion of small molecules. For membrane-permeant molecules (e.g., Hoechst 33342), the rate of interstitial penetration was extremely slow, compared to membrane-impermeant molecules (e.g., sodium fluorescein). This versatile tumor model could be applied to in vitro studies of transport and cellular uptake of drugs and genes.

Influence of osteopontin expression on the metastatic growth of CC531 rat colorectal carcinoma cells in rat liver

Development of hepatic metastasis is responsible for most of colorectal cancer-related deaths. Osteopontin (OPN) is a small integrin-binding N-linked glycoprotein, which plays a crucial role in the formation of hepatic metastasis. This study aimed to suppress Opn expression by an antisense-oligonucleotide (ASO(Opn)) to decrease liver metastasis in vivo. The effect of ASO(Opn) was investigated in vitro in CC531(lacZ) colorectal cancer cells in comparison to sense (SO) or nonsense (NSO) oligomers, by determining mRNA and protein expression levels, as well as cell survival. For in vivo treatment, CC531(lacZ) cells were intraportally inoculated into rats to compare the effects of ASO, SO and NSO oligomers, following prolonged subcutaneous administration by osmotic mini-pumps. The resulting CC531(lacZ) tumor cell load in the liver was measured by a β-galactosidase assay. Proliferation of CC531(lacZ) cells in vitro was significantly decreased after ASO(Opn) and SO treatment (P<0.001). Liver metastasis development was reduced as long as ASO(Opn) was administered, but this effect was rapidly blunted following the end of the ASO(Opn) administration. In contrast, administration of the SO resulted in a tumor load reduction, which surprisingly surpassed the ASO(Opn) effect in vivo in terms of a long-lasting metastasis suppression, which was accompanied with increased survival of the animals. Administration of the ASO(Opn) in rats was effective in decreasing their liver metastasis. The short-lived effect might be extended by modifications suited to increase the ASOs' half-life. In addition, there was a superior anti-metastatic effect caused by the SO, which has not been reported previously.

The influence of removal of primary tumor on incidence and phenotype of circulating tumor cells in primary breast cancer

Recent studies have shown that the detection of circulating tumor cells (CTC) pre and postoperatively in the peripheral blood of primary breast cancer patients may be an indicator for poor survival. This study aimed to investigate the influence of removal of the primary tumor on incidence and phenotype of circulating tumor cells in primary breast cancer. 209 primary breast cancer patients could be included into this analysis. Blood sampling was performed both pre and postoperatively. The blood specimens were immunomagnetically enriched using AdnaTest BreastCancerSelect within 4 h after blood withdrawal, followed by RNA isolation and subsequent gene expression analysis by reverse transcription and multiplex PCR using AdnaTest BreastCancerDetect. Three breast cancer-associated tumor markers and two hormone receptor genes were amplified: GA733-2, Muc-1, Her-2, ER, PR. In addition, bone marrow (BM) status was intraoperatively determined. Forty-three of 209 patients (21%) had pre and/or postoperatively circulating tumor cells. The positivity rates after surgery were higher but did not differ significantly (12% pre and 16% postoperatively, P = 0.264). Disseminated tumor cells in BM were seen in 32 of 209 cases (15%). Patients with positive BM status had significantly higher CTC positivity rates both pre and postoperatively compared to those with negative BM status. The most common CTC phenotype was triple negative (24 patients) followed by HER2+/ER-/PR- subtype (10) and ER and/or PR positive (9). Interestingly, 41 of 43 primary tumors (95%) were ER and PR positive. Removal of the primary tumor did not alter the phenotype of CTC. Surgery does not significantly influence the tumor cell load in the blood stream. CTC phenotype before and after the surgery generally remains identical but may differ from that of the primary tumor.

RIG-I Helicase-Independent Pathway in Sendai Virus-Activated Dendritic Cells Is Critical for Preventing Lung Metastasis of AT6.3 Prostate Cancer

We recently demonstrated highly efficient antitumor immunity against dermal tumors of B16F10 murine melanoma with the use of dendritic cells (DCs) activated by replication-competent, as well as nontransmissible-type, recombinant Sendai viruses (rSeV), and proposed a new concept, “immunostimulatory virotherapy,” for cancer immunotherapy. However, there has been little information on the efficacies of thismethod: 1) inmore clinically relevant situations including metastatic diseases, 2) on other tumor types and other animal species, and 3) on the related molecular/cellular mechanisms. In this study, therefore, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV on a rat model of lung metastasis using a highly malignant subline of Dunning R-3327 prostate cancer, AT6.3. rSeV/dF-green fluorescent protein (GFP)-activated bone marrow-derived DCs (rSeV/dF-GFP-DC), consistent with results previously observed in murine DCs. Vaccination of rSeV/dF-GFP-DC was highly effective at preventing lung metastasis after intravenous loading of R-3327 tumor cells, compared with the effects observed with immature DCs or lipopolysaccharide-activated DCs. Interestingly, neither CTL activity nor DC trafficking showed any apparent difference among groups. Notably, rSeV/dF-DCs expressing a dominant-negative mutant of retinoic acid-inducible gene I (RIG-I) (rSeV/dF-RIGIC-DC), an RNA helicase that recognizes the rSeV genome for inducing type I interferons, largely lost the expression of proinflammatory cytokines without any impairment of antitumor activity. These results indicate the essential role of RIG-I-independent signaling on antimetastatic effect induced by rSeV-activated DCs and may provide important insights to DC-based immunotherapy for advanced malignancies.

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients

Alloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use. We describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells. NK cells (6.0 ± 1.2 x 10(8)) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR+ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML. The approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.

The trifunctional antibody catumaxomab (anti-EpCAM × anti-CD3) in patients with malignant ascites: Immunomonitoring results of a pivotal phase II/III study (pooled population).

2521 Background: Malignant ascites (MA) is a symptom of late-stage tumor disease and associated with a poor prognosis. The trifunctional antibody catumaxomab is approved for treatment of MA. Methods: 258 EpCAM-positive ovarian (n=129) and nonovarian cancer (n=129) patients with MA were compared in an open-label, multicenter, randomized study for efficacy and tolerability of intraperitoneally administered catumaxomab. Various immunological and pharmacodynamical parameters were measured from ascites cell preparations. Results: In 170 patients randomized to catumaxomabtumor cells strongly decreased from a median of 6510 to 0 (after the last infusion). The in vivo target/effector ratio (EpCAM+/CD45+) showed a pronounced decrease from a baseline of 0.4404 at screening to 0.000 after the last infusion (all median). Leukocyte expansion was accompanied by in vivo upregulation of T-cell activation marker CD69 on CD4+ and CD8+ T cells in the MA samples. Concentration of the vascular endothelial growth factor (VEGF) was significantly decreased from screening (147 × 10-9) to after therapy (55 × 10-9) in MA samples (median; VEGF compared to total protein). Interestingly, at re-puncture visit catumaxomab patients (re- puncture at day 77, median) had a 9-fold lower and statistically significant (p=0.0012) tumor cell load compared to control patients (re-puncture at day 13, median). In vitro studies were carried out with screening samples to further validate the in vivo results. Similarly, the in vitro experiments showed EpCAM+ tumor cell elimination, leukocyte expansion, and activation of T cells. A strong upregulation of activating cytokines IL2, IL6, TNFα, and IFNγ (TH1 cytokine profile) was observed compared to control samples without catumaxomab. Conclusions: Intraperitoneal catumaxomab treatment triggers activation and proliferation of different types of immune cells and leads to long-lasting elimination of EpCAM+ tumor cells within malignant ascites. These data confirm the postulated trifunctional mode of action of catumaxomab in vivo and are in line with clinical efficacy in patients with MA. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Fresenius Biotech, TRION Research, TRION Pharma TRION Pharma TRION Pharma, TRION Research

Regional anaesthesia – the bride at last?

Regional anaesthesia – the bride at last?

PCR clonality detection in Hodgkin lymphoma

B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymphoma during follow-up of 1 to 6 years and no correlation was found between the presence of a clonal result and Epstein–Barr virus in the tumor cells. Our results indicate that the present routine PCR methods are sensitive enough to detect small numbers of malignant cells in cHL. Therefore, the presence of a clonal B-cell population does not differentiate between cHL and NHL.

Analysis of survival curve configuration is relevant for determining pathogenesis and causation

Improving technology helps us to identify more and more defects at the level of genes or proteins (event) as potential sources of a disease (effect), hopefully allowing more targeted cures with a "magic bullet". However, the complex interference of genes by the environment hinders the detection of strict causal relationships between defect and disease. We consider causality as temporal relationship between event and effect, thus causation is reflected by the configuration of "survival" curves. This is indicated by several survival curves of diseases with known causal relation. Furthermore, we discuss three theoretical models: a causal chain model, a causal field concept and a causal chain model with variable order, and present three assumptions about the specific consequences for configuration of outcome curves. Clinical examples of diseases that are caused by single hits reveal an S-shaped curve of cumulative incidence. In contrast, for diseases with numerous interacting pathogenetic effectors the superposition of all contributions results in widely linear cumulative incidence curves. The rare S-shaped deformation in the survival curves in patients with recurrent cancer is in conflict with our current view of recurrent cancer as mainly being a consequence of residual tumour cell load. The assumption of a "web of causation" instead of a "causal chain" reflects a more real situation for many clinical problems and can explain the widely seen absence of decisive, causally relevant conditions. As consequences for our current treatment of cancer is not insignificant, a careful analysis of the configuration of outcome curves with recognition of an S-shape may either help to identify causal therapies or may encourage more comprehensive approaches that consider the complexity of the disease.

Strong Impact of Micrometastatic Tumor Cell Load in Patients with Esophageal Carcinoma

To assess the role of immunohistochemically detectable nodal microinvolvement of patients with "curatively" resected esophageal carcinoma. In 73 patients with resectable esophageal carcinoma [squamous cell carcinoma (SCC), n = 45 (61.6%); adenocarcinoma (AC), n = 28 (38.4%)] a total of 2174 lymph nodes (LN) were removed. In each of the 1958 LN classified as negative on conventional histopathology, immunohistochemistry was performed using the anticytokeratin antibody AE1/AE3. To determine the role of the amount of residual tumor load, the patients were grouped according to the percentage of LN affected with micrometastasis (0%, <11%, and > or =11%). Tumor cells were immunohistochemically detected in 47 LN (2.4%) from 25 (34.2%) patients. Five-year overall survival probability (5-YSP) of 30% in pN(0 )patients with detected occult tumor cells in LN was significantly worse than that in those without nodal microinvolvement (76%, P = 0.021), hereby resembling that of pN1-patients (24%, P = 0.84). Median overall survival in patients with no (0%), low (<11%), and high (>11%) micrometastatic tumor load was 43, 27, and 11 months, respectively. Substratification according to histological type showed that, in patients with AC, the presence of nodal microinvolvement had a significant impact on 5-YSP (0% versus 65%; P = 0.03), whereas in patients with SCC, differences of 5-YSP were only of borderline significance (24% versus 53%; P = 0.081). Minimal tumor cell load as assessed by the ratio of micrometastatically affected LN is a complementary tool for better risk stratification of patients with esophageal carcinoma.

The diagnosis of Sézary syndrome on peripheral blood by flow cytometry requires the use of multiple markers

Diagnosis of Sézary syndrome (SS)-defining blood involvement is hampered by the lack of Sézary cell-specific markers and nonspecific morphology of the tumour cells. To identify the most reliable and easy to use markers for the diagnosis of SS-defining blood involvement. We studied 17 patients with SS and 11 control patients. We used flow cytometry for the detection of T-cell antigens (CD3, CD4, CD7 and CD8), expression of the Sézary cell-associated marker CD158k and T-cell receptor (TCR)-Vbeta chain. Additionally, Sézary cells were identified by peripheral blood smear for lymphocytes with cerebriform nuclei. It was not possible to diagnose blood involvement in all patients with SS by a single marker or method, although none of the markers was increased in the control population. Sézary cells were detected by blood smears in 13 of 17 (76%), by flow cytometry by their CD4+ CD7- CD3(dim) phenotype (> 1000 cells microL(-1)) in 13 of 17 (76%) and by expression of CD158k in 11 of 17 (65%) patients with SS. A specific T-cell clone was identified by identical TCR-Vbeta chain expression in 12 of 17 (71%) patients with SS. The identification of Sézary cells in individual patients varied for the different markers investigated. The combination of identifying CD4+ CD7- CD3(dim) cells, TCR-Vbeta chain and CD158k expression allowed a definite identification of SS-defining blood involvement in every individual patient. All of these markers can be measured by flow cytometry which would avoid time-consuming analysis of blood smears. These markers would also be suitable to monitor tumour cell load during therapy.

Abstract 3696A: The detection and prediction of circulating tumor cells in breast cancer patients

Abstract The detection of circulating tumor cells (CTC) in the blood of cancer patients is a promising tool for risk stratification, treatment tailoring, and monitoring of the disease state. The current study aims to develop a QPCR-based detection platform for the semi-quantitation of tumor cell load in the peripheral blood of breast cancer patients by combining gene expression data from 4 tumor marker genes into a single index-score. From a cohort of 131 individuals, CTC index-score positivity, indicating tumor cell presence, was observed in 14 out of 16 (88%) metastatic patients, 16 out of 87 (18%) non-metastatic stage I-III patients, and 0 out of 28 (0%) healthy female controls. A previous study demonstrated that CTC index-score positivity in metastatic patients correlated to a significantly shorter time to disease progression. This CTC detection platform demonstrates high specificity (100%) and sensitivity (88%), and can be an effective clinical tool providing valuable prognostic information. This CTC detection assay was subsequently compared to a CTC prediction assay developed using microarray expression data from the primary tumors of the patients from the same cohort. From 73 patients, a CTC predictive tumor profile was developed with the ability to distinguish those patients with a positive CTC QPCR index-score from those with a negative index-score with an average 83% internal cross-validation accuracy. When a small independent test set was assayed, those patients classified on the basis of their tumor gene expression as having a positive CTC QPCR score had a shorter interval to metastasis development (mean = 29.6 months) versus those predicted to have a negative score (mean = 51.4 months). When the QPCR-based and microarray-based CTC assays were compared directly, and each in combination with the “70-gene prognosis signature” for distant metastasis prediction, the CTC predictive microarray assay was superior to the CTC QPCR assay both alone and in combination with the 70-gene profile for time interval to metastasis. This study demonstrates that a microarray model predictive of tumor cell dissemination has prognostic value and seems independent of other metastasis-predictive microarray profiles.

Development of Immunogenic Tumor-Loaded Dendritic Cells Through Physical Perturbation and Apoptotic Cell Loading

To improve understanding of the forces that drive monocytes to transition into dendritic cells (), we developed an experimental system that converts monocytes to DC by passage of leukocytes through a 400 μm silica bead column. The results demonstrate that overnight culture of column-treated monocytes causes a phenotypic conversion that is characteristically displayed by immature DC. These phenotypic changes were enhanced when the DC were loaded with apoptotic cells, leading to increased expression of the DC maturation-associated markers CD83, CD80 and the chemokine receptor CCR7. The DC demonstrated potent induction of allogeneic T cell proliferation and the capacity to activate autologous CD8+ T cells. The CD8 T cells expressed augmented levels of perforin, IFN-γ and TNF-α and mediated CTCL cell apoptosis. These studies demonstrate that physical contact with silica beads combined with loading of apoptotic tumor cells induces synchronized, rapid conversion of human monocytes to DC, which can efficiently stimulate CD8+ T cells. These results may aid in the development of more efficient DC vaccines that can be loaded with the universe of antigens available in apoptotic tumor cells in a rapid, clinically practical fashion.

Relapse risk after autologous transplantation in patients with newly diagnosed myeloma is not related with infused tumor cell load and the outcome is not improved by CD34+ cell selection: long term follow-up of an EBMT phase III randomized study

This European Group for Blood and Marrow Transplantation (EBMT) multicentre randomized phase III study was designed to assess the safety and efficacy of CD34+ selection in newly diagnosed myeloma patients undergoing autologous transplantation. One hundred and eleven patients responsive to initial chemotherapy were randomized to receive CD34+ selected (arm A) or unselected PBPC (arm B) after conditioning with high-dose melphalan and TBI. ASO-PCR was used to assess purging efficacy and reinfused tumor load. Tumor load could be assessed in 59 patients. CD34+ selection gave a median tumor cell depletion of 2.2 logs (0.77-5.96). No tumor cells were detected in products infused in 17/26 (A) and 5/33 (B) patients. The five year overall survival (OS), event free survival (EFS) and relapse rate (RR) were 51%, 20% and 80% in arm A and 45%, 18% and 80% in arm B respectively with no significant difference between the two groups. Thirteen patients in arm A and 2 in arm B experienced episodes of serious early infection (p=0.02). There were 3 early transplant related deaths in A but none in B. Despite significant tumor cell reduction, CD34+ selection does not reduce RR and increases the risk of severe post-transplant infections. There was also no difference in RR between patients in either arm who received grafts with detectable tumor cells and those receiving grafts with no detectable tumor cells, suggesting that reinfused tumor cells may not be the main cause of relapse after autologous transplant in myeloma.

Treatment of ovarian cancer patients with malignant ascites using the trifunctional antibody catumaxomab: Results of a phase II/III study

5520 Background: Malignant ascites in ovarian carcinoma patients (pts.) is associated with a poor prognosis and reduced quality of life. Catumaxomab (anti-EpCAM × anti-CD3) is known to effectively eliminate tumor cells within ascites by simultaneously activating T cells and Fc gamma-receptor positive cells and redirecting them against the tumor. Methods: A total of 129 ovarian cancer pts. with recurrent symptomatic malignant ascites containing EpCAM+ tumor cells were enrolled in the study; 85 were randomized to treatment with catumaxomab (paracentesis plus intraperitoneal infusions of 10, 20, 50 and 150 μg for 11 days), and 44 to the control arm (paracentesis alone). The primary endpoint was puncture free survival (time to first need for paracentesis after treatment or time to death, which ever occurred first). Results: Pts. characteristics were well balanced in both arms. Median puncture free survival was 52 days for catumaxomab vs. 11 days for control (p&lt;0.0001) whereas the median time to first need for paracentesis was 71 days vs. 11 days l (p&lt;0.0001). There was a pronounced decrease of tumor cell load accompanied by a distinct increase of leukocyte count during catumaxomab treatment within the ascites fluid. Overall and progression free survival data suggest longer survival for catumaxomab-treated pts. compared to control. Follow-up data will be presented. The most frequent AEs were symptoms related to cytokine release (pyrexia, nausea, vomiting). These were generally mild to moderate in intensity, and fully reversible. Transient increases in liver enzymes and bilirubin, and transient WBC abnormalities such as leukocytosis, neutrophilia and a decrease in peripheral lymphocyte were regularly observed as abnormal laboratory values but rarely considered clinically significant. Conclusions: Intraperitoneal therapy with catumaxomab resulted in a significant and clinically relevant improvement of puncture-free survival time, tumor cell load, and time to first need for puncture compared to the control group of best available treatment. The safety profile reflects catumaxomabs mode of action and reveals a low and acceptable toxicity. No significant financial relationships to disclose.

A Pilot Study To Explore the Tolerability and Efficacy of Thalidomide Containing Regimens To Reduce Tumour Cell Load Prior to HSC in Multiple Myeloma and the Feasibility of Harvesting HSC Following Thalidomide Containing Regimens.

Aim: To explore the role of thalidomide in pre-transplant induction treatment in multiple myeloma.Patients and methods: Between Sept 2002 and March 2005, 37 patients with advanced, de-novo multiple myeloma (mean age 56 years, mean Serum. albumin 33g/L, and median b -2-microglobulin 4.0mg/L) were entered into a multicentre, phase 2 study of pre transplant induction treatment. The regimen included TDx3 (thalidomide 400mg/d, pulse dexamethasone 32mg TDS x 5d every 3 weeks PO), followed by DT-PACEx2 (thalidomide 400mg/d, dexamethasone 40mg/d x 4 PO and cisplatin 10mg/m2/d, doxorubicin 10mg/m2/d, cyclophosphamide 400mg/m2/day, etoposide 40mg/m2/d as 4 day infusion administered 4 weeks apart, supported with G-CSF 10m g/Kg/d). Thromboprophylaxis was warfarin (target INR 1.5–2.0) during TD and enoxaparin 40mg/day (adjusted according to platelet count) during DT-PACE. Stem cells were harvested at recovery from the second cycle of DT-PACE in the first 27 patients, but after review of the harvest results the remaining patients were harvested after first cycle of DT-PACE with an option to re-harvest after the second if the initial harvest was insufficient.Paraprotein and BJP responses and stem cell collections were compared to a historical cohort of 58 patients treated with VAD and mobilised with cyclophosphamide 5G/m2 and G-CSF 5m g/Kg.Results: 23/37 patients completed study treatment, 21 had successful stem cell harvests. There were 2 deaths (1 sepsis, 1 haemorrhage) and 2 failed stem cell harvests.After TD x 3 and VAD x3 the mean levels of paraprotein (or BJP) were 21% and 34% of pre-treatment levels, respectively (p=0.02). After DT-PACE x 2 and HD cyclophosphamide the mean levels of paraprotein (or BJP) were 14% and 31 % respectively (p=0.014).At the completion of TDx3 42% of patients had achieved VGPR and 50% PR, whereas after VAD x 3 there was 12% CR, 17% VGPR and 45% PR (p=0.231). Following DT-PACEx2 there was 22% CR, 39% VGPR and 26% PR and after HD cyclophosphamide there was 9% CR, 19% VGPR and 45% PR (p=0.039).The median number of CD34+ cells/kgBW harvested was 4.7 x 106 after DT-PACE and 11.6 x 106 after HD cyclophosphamide (p=0.001). The median number of aphereses procedures required was 2 for both study patients and historical controls. 58 serious adverse events included 20 episodes of infection (9 during TD and 11 during DT-PACE), 3 episodes of haemorrhage, 1 pulmonary embolus and 2 deaths.Conclusion:Thalidomide dexamethasone combination appears to be as efficacious as VAD in reducing tumour burden in de-novo multiple myeloma.The addition of DT-PACE improves the pre-transplant CR and VGPR rate.In most patients adequate stem cell harvest can be obtained, but yields appear to be less than after VAD/HD cyclophosphamide.Thalidomide dexamethasone followed by DT-PACE is associated with tolerable but not insignificant toxicity.

Combination treatment of CC531-lac-Z rat liver metastases by chemoembolization with pemetrexed disodium and gemcitabine

The aim of this study was to evaluate the combination effect of pemetrexed disodium (MTA; Alimta; LY 231514) and gemcitabine (GEM) administered by hepatic artery and portal vein chemoembolization (HACE and PVCE) in a colorectal cancer rat liver metastasis model. Proliferation studies on CC531-lac-Z rat colon cancer cells were performed using the MTT assay to obtain the optimal combination schedule of the two antineoplastic agents. To generate diffuse liver metastasis, 4 x 10(6) tumor cells were implanted into the portal vein of male WAG/Rij rats. MTA (30 mg/kg, 60 mg/kg, and 90 mg/kg) was administered locoregionally by portal vein chemoembolization (PVCE) and compared with repeated systemic intravenous injection. GEM (50 mg/kg) was also given locoregionally by hepatic artery chemoembolization (HACE) as well as systemically. All routes of administration were examined alone as well as in combination. Efficacy of treatment in terms of liver metastases burden was determined at the end of the experiment by measuring the beta-galactosidase activity of CC531-lac-Z cells with a chemoluminescence assay. Combination experiments in vitro showed a more than additive tumor cell reduction after sequential exposure to MTA preceding GEM (observed/expected ratio [O/E] = 0.73). Experiments with the reverse sequence (GEM-->MTA) resulted only in additive combination effects (O/E ratio = 1.08). Simultaneous drug exposure showed less than additive combination effects (O/E ratios > or = 1.25). In vivo, locoregional administration by HACE with GEM was significantly more effective than systemic intravenous bolus treatment (P = 0.03). Portal vein chemoembolization with MTA performed immediately after tumor cell inoculation was ineffective. Repeated systemic treatment with MTA yielded a slight reduction in tumor cell load that was significant versus control at the medium and high doses (60 mg/kg, P = 0.009; 90 mg/kg, P = 0.046) but not versus intraportal chemoembolization. The combination treatment of systemic (60 and 90 mg/kg) or locoregional (60 mg/kg) MTA with HACE using GEM (50 mg/kg) resulted in more than 80% tumor growth inhibition; this antineoplastic combination effect was maximally additive. A regimen-dependent synergistic combination effect of both drugs was found in vitro. In animals, hepatic artery chemoembolization with GEM was superior to systemic intravenous bolus treatment. Portal vein chemoembolization with MTA was ineffective. The optimal in vitro regimen of MTA (intravenous or PVCE) preceding GEM (HACE) resulted in a maximally additive tumor growth inhibition. The results indicate that MTA and GEM can successfully be combined and favor further evaluation in patients.

Chemoembolization of rat liver metastasis with microspheres and gemcitabine followed by evaluation of tumor cell load by chemiluminescence

An orthotopic, isogenic rat model was used to determine the potential of chemoembolization (CHE) for reducing the tumor cell load of a diffusely metastatic liver. Seven days after injecting CC531-lac-Z cells intraportally to male WAG/Rij rats, tumor positive animals were treated by injection into the hepatic artery with solvent (n=17), degradable starch microspheres (DSM, 30 mg/kg; n=16), DSM plus 5-fluorouracil (5-FU, dosages: 90, 60, and 40 mg/kg) or DSM plus gemcitabine (Gem, dosages: 100, 80, 50, and 10 mg/kg). After 3 more weeks the experiment was terminated, the livers were weighted and the number of CC531-lac-Z cells per liver was determined. Injection of DSM reduced the tumor cell load by 21% (T/C%=79), the combination with 5-FU caused a stimulation of growth at 40 mg/kg (T/C%=291; n=10), but effected dose-dependent reductions in tumor cell number at 60 mg/kg (T/C%=86; n=16), and 90 mg/kg (T/C=19; n=17). None of these effects was significantly different from controls. The combination of DSM plus Gem was toxic at the highest dose (100 mg/kg), but well tolerated and highly effective at 80 mg/kg (T/C%= 16; n=12), 50 mg/kg (T/C%=9; n=12), and 10 mg/kg (T/C%=26; n=14). These results were significantly different from controls (p<0.05), respectively. In summary, the comparison of CHE with 5-FU or Gem shows that the efficacy of Gem in reducing the hepatic tumor cell load was significantly higher and its therapeutic ratio was greater than that of 5-FU.

Significance of tumor necrosis factor- α and B2 microglobulin in patients having chronic lymphocytic leukemia

microglobulin (B2MG) is elevated in tumors, it is clinically used for lymphoproliferative diseases, where serum B2MG is related to tumor cell load, prognosis, and disease activity. The aim of this study was to investigate the serum levels of TNF-

Significance of tumor necrosis factor- α and B2 microglobulin in patients having chronic lymphocytic leukemia

Background and objectives: Tumor necrosis factor-α (TNF-α) is important for the growth and survival of the leukemic cells in B-cell chronic lymphocytic leukemia (B-CLL). B2 microglobulin (B2MG) is elevated in tumors, it is clinically used for lymphoproliferative diseases, where serum B2MG is related to tumor cell load, prognosis, and disease activity. The aim of this study was to investigate the serum levels of TNF-α and B2MG in B-CLL patients with correlation to relevant haematological data and disease characteristics. Patients, materials and methods: The study included 15 newly diagnosed untreated B-CLL patients obtained from the outpatient clinic at the National Cancer Institute in Cairo and 15 ages and sex matched controls. Venous blood samples were obtained from B-CLL and control groups for complete blood count (CBC). Serum was separated for measurement of TNF-α and B2MG levels by ELISA. Bone marrow (BM) aspiration was done to all B-CLL cases. Results: The studied B- CLL group consisted of 7 females and 8 males with mean ± SD age (54.9 ± 11.6 years). The clinical staging according to Rai classification was: 66.7% cases in stage 0-II and 33.3% cases in stage III-IV. There was highly significant elevation of white blood cell count (WBC), absolute lymphocytes in peripheral blood (PB), serum TNF-α and serum B2MG with high significant reduction of haemoglobin (Hb) in B-CLL group when compared with the control (P<0.001). There was significant reduction of platelets and significant elevation of absolute monocytes in PB in B-CLL group (P<0.01 & P<0.02 respectively). There was significant elevation of TNF- α in B-CLL patients with anaemia and B-CLL patients with thrombocytopenia in comparison with B-CLL patients without anaemia and B-CLL patients without thrombocytopenia (P=0.02 and P<0.05 respectively). There was highly significant positive correlation between TNF-α and both absolute monocytes in PB & serum B2MG with inverse highly significant correlation with Hb. A significant positive correlation was found between TNF- α and: WBC, BM lymphocytes and Rai III-IV disease stage with inverse significant correlation with platelets. A significant positive correlation was detected between B2MG and both WBC and absolute peripheral blood lymphocytes (P=0.01) with highly significant positive correlation with Rai III-IV disease stage (P<0.001), while no correlations were demonstrated with the other parameters. Conclusion: TNF-α and B2MG are important for the process of leukemogenesis and progression and may serve as bad prognostic markers for B- CLL. On the basis of these observations, therapeutic inhibition of TNF-α and B2MG could be a new strategy of importance in the treatment of B-CLL.