Tumor Cell Burden Research Articles

Neoadjuvant treatment of melanoma: case reports and review

Neoadjuvant therapy is an under-utilized regimen for the treatment of metastatic melanoma. The use of this approach has been increasing in other tumor types. Neoadjuvant therapy may reduce occult circulating tumor cell burden in the face of bulky disease and afford a real time evaluation of treatment effectiveness. Neoadjuvant approach can also provide preoperative histologic and molecular analysis of treated tissue that may guide the postoperative treatment planning in patients with resectable metastatic melanoma lesions. The putative benefits of better margin control and clearance of occult systemic disease would theoretically improve surgical outcome. With the advent of effective agents against metastatic melanoma, this common approach to the treatment of rectal cancer, metastatic colon cancer, and breast cancer should also be evaluated as a viable treatment strategy for advanced stage melanoma.

PLASMOCITOMA CUTÂNEO METASTÁTICO EM DOENTE COM MIELOMA MÚLTIPLO

Introdução: No mieloma múltiplo as lesões cutâneas são raras e ocorrem em fases tardias da doença, como um reflexo da elevada carga tumoral. Caso clínico: Os autores relatam o caso de uma doente de 66 anos com mieloma múltiplo IgG lambda que, após 16 meses de evolução e aparentemente sob controlo hematológico, desenvolveu uma placa eritemato-violácea, dolorosa e de consistência dura, atingindo a face posterior da coxa direita. A biopsia cutânea confirmou o diagnóstico de plasmocitoma cutâneo. Radioterapia local induziu regressão completa da lesão cutânea, no entanto a doente faleceu 4 meses depois. Discussão: O plasmocitoma cutâneo secundário surge habitualmente por contiguidade a partir de lesões ósseas subjacentes, manifesta-se por placas ou nódulos eritemato-violáceos infiltrados, são extremamente raros e indicam um prognóstico desfavorável como no nosso caso. A apresentação da lesão como erisipela-like sem doença óssea subjacente é ainda mais raro.PALAVRAS-CHAVE – Mieloma múltiplo; Plasmacitoma; Neoplasias da pele.

Quantification of BCR-ABL mRNA in Plasma/Serum of Patients with Chronic Myelogenous Leukemia

Quantification of tumor-associated mRNA extracted from blood cells/tissues containing tumor cells is used for evaluation of treatment efficacy or residual tumor cell burden in tumors including leukemia. However, this method using tumor cell-containing blood/tissue is difficult to evaluate the whole tumor cell burden in the body. In order to establish an efficient method to evaluate the whole tumor cell burden in the body, we tried to quantify tumor-associated mRNA existing in plasma/serum instead of leukemia cell-containing blood cells in patients with chronic myelogenous leukemia (CML) and compared the levels of BCR-ABL mRNA between plasma/serum and peripheral blood cells. mRNA of BCR-ABL, WT1 or GAPDH (control molecule) was detected by real-time RT-PCR using RNA extracted from plasma/serum of almost all the patients with CML. Copy numbers of BCR-ABL mRNA were significantly correlated between plasma/serum and peripheral blood cells. However, levels of BCR-ABL mRNA extracted from serum were low compared with those extracted with peripheral blood cells. The present findings suggest that although real-time RT-PCR of mRNA existing in plasma/serum could be used for evaluating the whole tumor cell burden in the body, it's required to establish an efficient method to quantify plasma/serum mRNA by nature without degrading during the procedure.

Immunoglobulin light chain, Blimp-1 and cytochrome P4501B1 peptides as potential vaccines for AL amyloidosis.

Amyloid light chain (AL) amyloidosis is a lethal disorder characterized by the pathologic deposition of clonal plasma cell-derived, fibrillogenic immunoglobulin light chains in vital organs. Current chemotherapeutic regimens are problematic in patients with compromised organ function and are not effective for all patients. Here, a platform of computer-based prediction and preclinical mouse modeling was used to begin development of a complementary, immunotherapeutic approach for AL amyloidosis. Three peptide/MHC I-binding algorithms identified immunogenic peptides from three AL plasma cell-associated proteins: (1) amyloidogenic λ6 light chains, (2) CYP1B1, a universal tumor antigen hyper-expressed in AL plasma cells and (3) B lymphocyte-induced maturation protein 1 (Blimp-1), a transcription factor required for plasma cell differentiation. The algorithms correctly predicted HLA-A(*)0201-binding native and heteroclitic peptides. In HLA-A2 transgenic mice, these peptides, given individually or in combination, induced potent CTL which kill peptide-loaded human lymphoma cells and/or lymphoma cells producing target protein. Blimp-1 peptide-immunized mice exhibited a reduced percentage of splenic, lymph node and bone marrow plasma cells and a decrease in the absolute number of splenic plasma cells demonstrating (1) presentation of target peptide by endogenous plasma cells and (2) appropriate CTL homing to lymphoid organs followed by killing of target plasma cells. These studies suggest that AL amyloidosis, with its relatively low tumor cell burden, may be an attractive target for peptide-based multivalent vaccines.

Targeted Removal of Migratory Tumor Cells by Functionalized Magnetic Nanoparticles Impedes Metastasis and Tumor Progression

To slow tumor progression by reducing migratory tumor cell burden using magnetic nanoparticles (MNPs) functionalized with ligands selective for malignant cell surface receptors. Three groups of female C57BL/6 mice (control group I, control group II and experimental group) were intraperitoneally injected with a murine ovarian cancer cell line (ID8[VEGF160(+)/eGFP(+)]). Control group I received no intervention. MNPs were functionalized with ephrin-A1 mimetic peptides selective for the EphA2 receptor that is highly expressed by several cancers. Peritoneal fluids were removed by paracentesis from the experimental group and mixed with the functionalized MNPs. Magnetic filtration was used to remove particle/malignant cell conjugates and filtered peritoneal fluids were re-introduced intraperitoneally. Control group II received the same treatment as the experimental group without MNPs. Experimental group tumor progression was 10.77-times slower than that of control group I. Reduction of malignant cell titer significantly prolonged time to end point in a metastatic ovarian cancer model.

Mena deficiency delays tumor progression and decreases metastasis in polyoma middle-T transgenic mouse mammary tumors

IntroductionThe actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development.MethodsTo investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice.ResultsMena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching.ConclusionsDeficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena deficiency during development causes defects in invasive processes involved in mammary gland development. These findings suggest that functional intervention targeting Mena in breast cancer patients may provide a valuable treatment option to delay tumor progression and decrease invasion and metastatic spread leading to an improved prognostic outcome.

Aggressive NK Cell Leukemia (ANKL): Experience with 34 Patients and Therapeutic Potentials of L-Asparaginase and Allogeneic Hematopoietic Cell Transplantation - A Japan-Korea Multicenter Study for ANKL (ANKL07) -

AbstractAbstract 3091 Background:Aggressive NK cell leukemia (ANKL) is a malignant disorder of mature NK cells that is more common in East Asia and is often associated with Epstein-Barr virus (EBV). The prognosis of ANKL is dismal and the survival is one of the shortest among the lymphoid neoplasms. Because of its rarity, the characteristics and optimal managements of this disease are uncertain.To better elucidate clinicopathologic features and therapeutic modalities for ANKL, a retrospective Japan-Korea multicenter survey of ANKL (ANKL07) was conducted. Methods:Eligibility criteria were based on the diagnosis for ANKL according to the WHO classification. Pathological and clinical information including that on chemotherapy and hematopoietic cell transplantation (HCT) was collected. Approval from the institutional review board at each participating institution was obtained for this study. Results:Forty-one patients (pts) were registered from 8 hospitals. After central reviews, 34 pts were further analyzed, while 7 pts were excluded.The median age of the pts was 45 years old (range: 16–79) with 26 males and 8 females. Four pts had a history of preceding disorders including EBV-LPD in childhood, mosquito bite hypersensitivity or EBV-associated liver damage. Three female pts presented during pregnancy. Fever and hepatosplenomegaly were recognized in 100% and 71% of the pts, respectively. Thirty-two pts (94%) showed high or high-intermediate International Prognosis Index. Three types of ANKL cells (type I, II or III) were categorized according to the morphological features. Using this classification, there were 11, 13 and 10 pts with types I, II and III, respectively. EBV was positive in 85% of the pts. Eleven pts showed less than 20% leukemic cells in both peripheral blood (PB) and bone marrow (BM). The clinical characteristics and prognosis of these pts did not differ from those of the pts with more tumor cells in PB/BM except for the incidence of hemophagocytic syndrome.As initial therapy, anthracycline-based chemotherapies were utilized in 13 pts and L-asparaginase-containing regimens were administered in 5 pts. Although anthracycline chemotherapy was not associated with better outcome by Kaplan-Meier analysis (p=0.64), L-asparaginase chemotherapy resulted in CR or PR in 4 pts and significantly better survival (p=0.03).A total of 9 HCT, 2 autologous (auto) and 7 allogeneic (allo), were performed in 8 pts; none were in complete remission (CR) at the time of transplant. For allo HCT, the donor source was HLA-matched related bone marrow in two, cord blood in two, PB of HLA-matched unrelated donor in one and HLA-mismatched related PB in two. The conditioning regimen was TBI+CY in three, and fluradabine-based regimen in four. One patient with auto HCT and 4 with allo HCT reached CR after HCT.The median survival of all 34 pts was 51 days (range: 1-1, 630). Median survivals for the patients with or without HCT were 266 days (range: 80-1, 630) and 36 days (range: 1–324), respectively. Two pts with allo HCT were alive in CR while the other 6 pts with allo HCT died of the disease (5 pts) or infection (1 pt). All pts without HCT died of ANKL. Times from the diagnosis to HCT for the 2 survivors were within 80 days.In univariate analysis, L-asparaginase administration was the only clinical factor associated with better survival (hazard ratio 0.29, 95% confidence interval: 0.12–0.70, p=0.008). Age (HR 1.02, 95% CI: 0.998–1.08, p=0.07) and the use of etoposide (HR 0.48, 95% CI: 0.22–1.07, p=0.07) were marginal. Multivariate Cox analysis indicated that the use of L-asparaginase was an independently significant factor for better survival (HR 0.33, 95% CI: 0.13–0.83, p=0.02). Conclusions:This is the largest case series of ANKL. ANKL cells are morphologically heterogeneous and the main infiltration sites of ANKL are liver, spleen and BM. In addition, ANKL showed poor prognosis regardless of PB/BM tumor cell burden. Although the prognosis of ANKL did not improve significantly compared with those in previous reports, L-asparaginase-containing initial chemotherapy and allo HCT are promising approaches for ANKL. Prospective studies are required to confirm these results. Disclosures:No relevant conflicts of interest to declare.

Consolidation with Bortezomib, Thalidomide and Dexamethasone After High Dose Therapy Is Feasible, Safe and Effective In De Novo Multiple Myeloma Patients Who Already Received New Drugs Containing-Induction Regimen

AbstractAbstract 3041High dose therapy (HDT) with autologous stem cell transplantation (ASCT) is a standard treatment option for frontline myeloma patients (pts). However, after a single or a double transplantation, almost all pts ultimately relapse. Thus, new strategies are required to control the residual disease after HDT. Consolidation therapy, given early after HDT, could enhance the depth of response and further improve progression free survival (PFS) and overall survival (OS). The IFM (Intergroupe Francophone du MyeÃŒlome) previously reported that either Thalidomide or Lenalidomide, administered after HDT, was able to reduce this residual disease (IFM 99-02 and 2005-02 trials, Attal M et al. Blood 2006 and ASCO 2010). Ladetto M et al. (JCO 2010) also reported a major shrinking of residual tumor cell burden in myeloma pts undergoing early consolidation with Bortezomib, Thalidomide and Dexamethasone (VTD) after ASCT. None of these pts had previously received treatment with Thalidomide and/or Bortezomib during induction. The aim of this study was to evaluate the feasibility, safety and efficacy of early consolidation therapy in pts who had received new drugs containing-induction therapies before ASCT. Patients and Methods:In this prospective monocenter study,patients were eligible if they had the following: 1) at least partial response (PR) after HD melphalan (HDM), 2) no grade ≥ 2 peripheral neuropathy (PNY). The consolidation therapy with vTD had to be started within 3 months from ASCT for a total of 2 cycles. Each 4-week cycle consisted of: a) Bortezomib: 1mg/m2 as an IV injection twice weekly (on days 1, 4, 8, 11); b) oral Thalidomide: 100 mg/day; c) oral Dexamethasone 40 mg/day once a week (on days 1, 8, 15 and 22). Pts did not receive maintenance therapy after vTD consolidation. A systematic prophylactic anticoagulation therapy was given either by aspirin or low molecular weight heparin. Pts also received systematic anti herpes zoster prophylaxis with valacyclovir. Toxicities were graded according to the CTAECv4 at each cycle. Response was assessed according to modified European group for Blood and Marrow Transplantion criteria including very good partial response (VGPR), 1 month after the last cycle of vTD. As no immunophenotypic analysis was available routinely in our institute, we did not evaluate stingent complete response. Results:From August 2008 to May 2010, 90 newly diagnosed multiple myeloma pts under 65 received HDM followed by ASCT in the Toulouse's hospital bone marrow transplant unit, FRANCE. Forty-six pts were eligible for this study and started on consolidation. Two pts are ongoing their treatment and 41 received the whole planned treatment. Two pts withdrawn thalidomide and 1 pt did not receive the second cycle of consolidation because of toxicities. Forty-four pts were excluded mainly because of PN (27%). Initial characteristics were: age=58 years (range,44-65); ISS: 1= 50%, 2= 23%, 3=18%, NA= 9%; DS stage: I=4,5%, II=4,5%, III=91%; median beta-2microglobulin=2,9 mg/l (1,3-11,3); FISH analysis: t(4-14) and/or del 17p= 4/29 evaluable pts. The induction regimen was: Bortezomib/Dexamethasone (82%), vTD (11%), or another regimen (7%).Before consolidation therapy, response rates were: PR=16%, VGPR=48%, near complete response (nCR)=14% and CR=23%. In an Intend to treat basis, 17 pts (39%) improved their responses after consolidation with vTD: 3 pts (7%) from PR to VGPR, 8 pts (18%) from VGPR to nCR, and 6 pts (14%) from VGPR to CR. Response improvement was also reported in the 5 pts who already received induction therapy with vTD; three pts (60%) improved their response: 1 pt from PR to VGPR and 2 pts from VGPR to nCR. As immunophenotypic analysis was not avalaible, response improvement might be underestimate for CR pts. Overall, consolidation therapy with 2 cycles of vTD was efficient; 16 pts (36%) acheived CR, 30 pts (68%) CR+nCR and 40 pts (91%) VGPR or better.Considering the safety profile, there was no toxic death or grade 3/4 hematological toxicity. Non-hematological toxicities reported were: fatigue=18%, PNY (all grades)= 9%, pneumonia=7%, vertigo= 7%, constipation= 6%, and deep-vein thrombosis= 4,5%. Conclusions:Early consolidation with vTD in pts who already received Thalidomide and/or Bortezomib as induction therapy is feasible, safe and effective. Response rates improved in almost 40% of pts. Disclosures:Roussel:Janssen: Consultancy, Research Funding, orator; celgene: Consultancy, Orator, Research Funding. Off Label Use: bortezomib and thalidomide as consolidation therapy after High Dose Melphalan. Attal:celgene: Consultancy, Research Funding; janssen: Consultancy, Research Funding.

Efficacy of LY2784544, a Small Molecule Inhibitor Selective for Mutant JAK2 Kinase, In JAK2 V617F-Induced Hematologic Malignancy Models

AbstractAbstract 4087 Introduction:The JAK2-V617F mutation is the most common molecular abnormality in the BCR/ABL-negative myeloid proliferative neoplasms (MPNs) and is present in approximately 95% of patients with polycythemia vera (PV) and roughly 60% of patients with either essential thrombocythemia (ET) or primary myelofibrosis (PMF). The JAK2 V617F kinase is constitutively active, oncogenic and recapitulates MPN in murine models. Therefore, mutant JAK2 is a potential therapeutic target for MPNs. Here, we describe the efficacy of the clinical candidate LY2784544; a novel small molecule inhibitor selective for JAK2 V617F mutant, in JAK2 V617F-induced in vitro and in vivo models. Methods:Assessment of the ability of LY2784544 to inhibit the signaling of the V617F mutant or wild type JAK2 was evaluated in Ba/F3 cells expressing V617F or wild type JAK2, whereby the cellular level of phospho-STAT5 (pSTAT5) was measured by a high content imaging (Cellomics) assay. The antiproliferative activity of LY2784544 and its ability to induce apoptosis were examined by Celltiter-Glo Cell Viability and Caspase-Glo 3/7 Apoptosis Assays, respectively. The effect of LY2784544 on JAK3 was investigated in IL-2 stimulated NK-92 (nature killer) cells by measuring the inhibition of JAK3-STAT5 signaling with the Cellomics assay. To examine the in vivo target inhibition, a JAK2 V617F-induced ascites tumor model was established by implanting Ba/F3-JAK2 V617F-GFP cells into the intraperitoneal cavity of severe combined immunodeficiency (SCID) mice. After orally administering LY2784544, the inhibition of pSTAT5 was measured in Ba/F3-JAK2 V617F-GFP ascites tumor cells by fluorescence activated cell sorting (FACS) assay. The anti-tumor efficacy of LY2784544 was investigated in a SCID mouse model of MPN developed by intravenously implanting Ba/F3-JAK2 V617F-GFP cells. After oral treatment for 7 or 14 days, twice daily (BID), tumor burden reduction and the effect on erythroid progenitor cells were determined by measuring the GFP positive tumor cells and CD71/Ter119 positive cells in the spleens with FACS assays, respectively. Results:In the in vitro tests using Ba/F3 cells expressing either wild type or V617F mutant JAK2, LY2784544 potently inhibited the JAK2 V617F-STAT5 signaling at a concentration that was 41-fold lower than that required to inhibit IL-3-activated wild type JAK2-STAT5 signaling (IC50=0.055 μ M for JAK2 V617F vs. 2.26 μ M for WT JAK2). Similarly in the proliferation assay, LY2784544 selectively inhibited the JAK2 V617F-driven cell proliferation (IC50= 0.068 μ M). Inhibition of JAK2 V617F signaling correlated well with the induction of apoptosis (EC50= 0.113 μ M) in Ba/F3 cell model. Consistent with the observed apparent selectivity for the V617F mutant and reduced sensitivity against wild type JAK2, a lower potency was observed with LY2784544 in the IL-3-dependent cell proliferation assay (IC50=1.356 μ M) as well as less effective inhibition of IL-2 dependent JAK3-STAT5 signaling in NK-92 cells (IC50=0.94 μ M). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2 V617F-GFP ascites tumor cells with a Threshold Effective Dose 50 (TED50) of 12.7 mg/kg. Consistent with this effective inhibition of JAK2V617F, LY2784544 also significantly reduced Ba/F3-JAK2 V617F-GFP tumor burden in the JAK2 V617F-induced MPN model with a TED50 of 13.7 mg/kg after oral treatment (BID) for 14 days, indicating that the efficacy of LY2784544 is mediated by pharmacological inhibition of JAK2 V16F-STAT5 pathway. Furthermore, LY2784544 selectively reduced Ba/F3-JAK2 V617F-GFP tumor cells with no effect on CD71/Ter119 positive erythroid progenitors in spleens of SCID mice after oral treatment (BID) for seven days. Conclusions:The in vitro data suggest that constitutively active JAK2 V617F is more sensitive to LY2784544 than cytokine-activated wild type JAK2. Consistent with this observation, in MPN disease model testing, LY2784544 selectively reduces JAK2 V617F tumor cell burden with no effect on erythroid progenitor cells. These findings suggest that as a JAK2 V617F selective targeted therapy, LY2784544 has the potential to induce apoptosis in JAK2 V617F malignant clones while potentially minimizing unintended effects on the normal hematopoietic progenitor cells. These results helped to support the advancement of LY2784544 into an ongoing phase I trial in the MPNs (I3X-MC-JHTA, NCT01134120). Disclosures:Ma:Eli Lilly and Company: Employment. Zhao:Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment. Clayton:Eli Lilly and Company: Employment. Blosser:Eli Lilly and Company: Employment. Burkholder:Eli Lilly and Company: Employment. Smith:Eli Lilly and Company: Employment.

Inhibition of KSHV-associated lymphoma engraftment in SCID mouse by morpholino oligomers

Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is associated with several malignant disorders, including Kaposi’ ss arcoma, primary effusion lymphoma (PEL), and multicentric Castleman’s disease. We have explored peptide-conjugated antisense phosphorodiamidate morpholino oligonucleotides (PPMOs) against KSHV and found effective PPMOs in inhibition of KSHV gene expression in cell culture. PPMOs are single-stranded DNA analogues that have a modified backbone and penetrate cells readily. In this study, we further tested the PPMOs in a SCID mouse model to assess their effect on engraftment and growth of PEL cells. PEL cells were engrafted into SCID mice via intraperitoneal route. PPMO was administered at the same time and repeated every other day for 10 doses. The mice were observed and scored for ascites development. The tumor cell burden was assessed by flow cytometry. Administration of anti-vIL-6 PPMO protected the mice from lymphoma development, while those mice receiving a control PPMO developed ascites and had high ratio of PEL cells in peritoneal lavage. The results demonstrate that PPMO against key KSHV genes can potently reduce KSHV replication and growth of PEL cells in SCID mice. Further exploration of PPMOs in the animal model is warranted.

Pharmacokinetics of BSI-201, a poly (ADP-ribose) polymerase-1 (PARP1) inhibitor, in cerebrospinal fluid (CSF) of a patient with breast cancer with carcinomatous meningitis.

e13559 Background: Triple-negative breast cancer (TNBC) carries a high risk for brain metastasis and is associated with poor survival. A key role has emerged for PARP1 in the repair of DNA damage, particularly in cancers associated with BRCA1 dysfunction, including TNBC. BSI-201, a small molecule inhibitor of PARP1, has demonstrated significant antitumor activity when added to gemcitabine and carboplatin in treatment of metastatic TNBC. BSI- 201 was provided through a single patient IND to a patient with metastatic TNBC who developed carcinomatous meningitis as the only site of initial relapse. Methods: The patient had an Omaya reservoir inserted to facilitate intrathecal (IT) chemotherapy. The patient received IT methotrexate, which was switched to liposomal-cytarabine due to arachnoiditis. The patient received BSI-201 (5.6 mg/kg IV, biweekly) throughout IT treatment. CSF was sampled via the Omaya reservoir, and BSI-201 and its metabolites were measured prior to dosing and up to 2 hours post-infusion. Results: BSI-201 exposure in the CSF was ∼120 ng/mL at end-of-infusion. BSI-201 was rapidly eliminated from the CSF (t1/2 ∼ 10 min), with a longer-lived metabolite (t1/2 ∼ 21min). Overall exposure of the CNS was estimated to be ∼8% of the total systemic AUC for BSI-201, and Cmax in CSF was ∼7.5% of plasma Cmax. The patient responded to treatment for 5 months before developing disease progression in the bone and a second primary breast cancer. The burden of tumor cells in the CSF was reduced by > 95% throughout treatment. No evidence of systemic or neurologic toxicity from BSI-201 beyond what is normally observed with chemotherapy occurred. Conclusions: BSI-201 and its key metabolites cross the blood-brain barrier with BSI-201 AUC and Cmax in CSF of ∼10% of plasma values. This observation provides a basis for further examination of the role of BSI-201 in the treatment of this metastatic complication. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration BiPar Sciences

Monitoring of Minimal Residual Disease (MRD) after Multidrug Chemotherapy and Its Correlation to Outcome in Dogs with Lymphoma: A Proof-of-Concept Pilot Study

Tumor cell burden in dogs with lymphoma cannot be assessed accurately by diagnostic evaluation during clinical complete remission (CR). Recent advances in polymerase chain reaction (PCR)-based methods enabled us to quantify minimal residual disease (MRD) in canine lymphoma. To quantify MRD in dogs with lymphoma treated with multidrug chemotherapy and to correlate it with remission duration after chemotherapy. Seventeen dogs with lymphoma that achieved CR by multidrug chemotherapy. Rearranged immunoglobulin heavy chain or T-cell receptor gamma chain gene fragments from lymphoma cells were PCR amplified and sequenced to prepare clone-specific primers and probes for real-time PCR to quantify MRD. MRD in the peripheral blood was monitored during and at the end of a 25-week multidrug chemotherapy protocol. Correlation between MRD at the end of chemotherapy and remission duration after chemotherapy was analyzed. MRD gradually decreased after initiation of multidrug chemotherapy, reached a nadir as low as <0.019-1.0 cells/microL at weeks 4-17, and remained low or slightly increased until week 25. MRD at the end of chemotherapy was negatively correlated with remission duration from the end of chemotherapy to relapse. MRD could be an objective marker to indicate tumor cell burden in dogs with lymphoma even in clinical CR. MRD at the end of chemotherapy could be a prognostic factor to predict remission duration after chemotherapy.

Quantification of tumor cell burden by analysis of single cell lymph node disaggregates in metastatic prostate cancer

The size of lymph node (LN) metastases in prostate cancer patients represents an important prognosticator, but histological work-up may not reflect the true extent of tumor invasion. We present a novel technique (1) to detect early tumor cell dissemination and (2) to quantify the true tumor burden. Prospectively 232 LN of 20 consecutive patients with prostate cancer after lymph node dissection were longitudinally bisected, one half was subjected to single cell immunocytochemistry for pancytokeratine (CK), the other half underwent routine histopathological work-up and step section analysis. In immunocytochemistry, tumor cell density (TCD) was quantified by calculating the number of CK-positive cells/million leucocytes and compared to routine histopathology and step section analysis. Eight of 20 patients were positive in histopathology and step sectioning, but 14 of 20 patients were positive in single cell analysis. Twenty-five of 232 LN were positive in routine histopathology, whereas 52 of 232 LN were positive in single cell analysis. Median TCD in histopathologically positive LN was 3060.0 x 10(-6) and 9.9 x 10(-6) in histopathologically negative LN (P < 0.0001). Mean TCD of histopathologically negative LN of pN1 patients was significantly higher than the mean TCD of pN0 patients (P < 0.003). Mean TCD per patient correlated with serum-PSA (r(2) = 0.48, P < 0.006). Single cell analysis has an increased detection rate compared to routine histopathology and even to serial step section analysis. The method can detect early tumor dissemination and enables quantification of the tumor burden. The subgroup of histopathologically negative LN with CK-positive cells represents tumor cell dissemination not depicted histologically.

Cytotoxicity and antiangiogenic activity of grandisin

Abstract Objectives The antitumoural properties of grandisin, a tetrahydrofuran neolignan from Piper solmsianum, were investigated by in-vitro and in-vivo assays using the Ehrlich ascites tumoural (EAT) model. Methods Viability of the tumour cells was evaluated by Trypan blue exclusion and MTT methods, after incubation with grandisin (0.017-2.3 μM). The effects of grandisin on the activity of caspase-3, −6, −8, and −9 were also investigated using colorimetric protease kits. In-vivo studies were performed in EAT-bearing mice treated intraperitoneally with 2.5, 5 or 10 mg/kg grandisin for 10 days. Key findings Grandisin inhibited the growth of EAT cells, by both methods, with IC50 values less than 0.25 μM. The results showed that the activity of all the caspases studied increased in grandisin-treated cells, when compared with control, non-treated cells. Administering grandisin to EAT-bearing mice increased survival of the animals, in a dose-dependent manner. Simultaneously, we detected a 66.35% reduction of intraperitoneal tumour cell burden in the animals treated with 10 mg/kg grandisin. Additionally, in these animals, the marked increase of vascular endothelial growth factor (VEGF) levels, induced by EAT development, was decreased with treatment with grandisin, resulting in a reduction of 32.1% of VEGF levels in the peritoneal washing supernatant, when compared with the control. Conclusions The results demonstrated that grandisin induced in-vitro cytotoxicity and antiangiogenic effects in mice while it acted against tumour evolution, prolonging host survival.

Imaging features of invasion and preoperative and postoperative tumor burden in previously untreated glioblastoma: Correlation with survival

Background:A paucity of data exists concerning the prognostic usefulness of preoperative and postoperative imaging after resection of glioblastoma multiforme (GBM). This study aimed to connect outcome with imaging features of GBM.Methods:Retrospective computer-assisted volumetric calculations quantified central necrotic (T0), gadolinium-enhanced (T1) and increased T2-weighted signal volumes (T2) in 70 patients with untreated GBM. Clinical and treatment data, including extent of resection (EOR), were obtained through chart review. T1 volume was used as a measure of solid tumor burden; and T2 volume, as an indicator of invasive isolated tumor cell (ITC) burden. Indicators of invasiveness included T2:T1 ratios as a propensity for ITC infiltration compared to solid tumor volumes and qualitative analysis of subependymal growth and infiltration of the basal ganglia, corpus callosum or brainstem. Cox multivariate analysis (CMVA) was used to identify significant associations between imaging features and survival.Results:In the 70 patients studied, significant associations with reduced survival existed for gadolinium-enhancing tumor crossing the corpus callosum (odds ratio, 3.14) and with increased survival with gross total resection (GTR) (GTR median survival, 62 weeks versus 37 and 34 weeks for sub-total resection and biopsy, respectively). For a selected “GTR-eligible” subgroup of 52 patients, prolonged survival was associated with smaller preoperative gadolinium-enhancing volume (T1) and actual GTR.Conclusion:Some magnetic resonance (MR) imaging indicators of tumor invasiveness (gadolinium-enhancing tumor crossing the corpus callosum) and tumor burden (GTR and preoperative T1 volume in GTR-eligible subgroup) correlate with survival. However, ITC-infiltrative tumor burden (T2 volume) and “propensity” for ITC invasiveness (T2:T1 ratio) did not impact survival. These results indicate that while the ITC component is the ultimate barrier to cure for GBM, the pattern of spread and volumes of gadolinium-enhancing solid tumor are more robust indicators of prognosis.

Cytotoxicity and antiangiogenic activity of grandisin

The antitumoural properties of grandisin, a tetrahydrofuran neolignan from Piper solmsianum, were investigated by in-vitro and in-vivo assays using the Ehrlich ascites tumoural (EAT) model. Viability of the tumour cells was evaluated by Trypan blue exclusion and MTT methods, after incubation with grandisin (0.017-2.3 microm). The effects of grandisin on the activity of caspase-3, -6, -8, and -9 were also investigated using colorimetric protease kits. In-vivo studies were performed in EAT-bearing mice treated intraperitoneally with 2.5, 5 or 10 mg/kg grandisin for 10 days. Grandisin inhibited the growth of EAT cells, by both methods, with IC50 values less than 0.25 microm. The results showed that the activity of all the caspases studied increased in grandisin-treated cells, when compared with control, non-treated cells. Administering grandisin to EAT-bearing mice increased survival of the animals, in a dose-dependent manner. Simultaneously, we detected a 66.35% reduction of intraperitoneal tumour cell burden in the animals treated with 10 mg/kg grandisin. Additionally, in these animals, the marked increase of vascular endothelial growth factor (VEGF) levels, induced by EAT development, was decreased with treatment with grandisin, resulting in a reduction of 32.1% of VEGF levels in the peritoneal washing supernatant, when compared with the control. The results demonstrated that grandisin induced in-vitro cytotoxicity and antiangiogenic effects in mice while it acted against tumour evolution, prolonging host survival.

The Urokinase Receptor Promotes Cancer Metastasis Independently of Urokinase-Type Plasminogen Activator in Mice

The urokinase receptor (uPAR) promotes metastasis of human malignancies; however, its mechanism of action remains incompletely understood. Established models focus on the ability of uPAR to bind urokinase-type plasminogen activator (uPA) and promote protease activation in the tumor cell microenvironment; however, uPAR also regulates cell signaling and migration by both uPA-dependent and -independent mechanisms in vitro. The significance of uPAR as a cell-signaling receptor in vivo remains unclear. In this study, we expressed either human or mouse uPAR in human embryonic kidney (HEK-293) cells. We selected HEK-293 cells because, unlike most cancer cells, they do not express uPA or uPAR endogenously. Both mouse and human uPAR increased cell adhesion and migration on vitronectin. Rac1 was activated and responsible for the increase in cell migration. HEK-293 cells that did not express uPAR formed palpable tumors in severe combined immunodeficient mice; however, metastases were exceedingly rare. The xenografts contained abundant mouse uPA, produced by infiltrating mouse cells, but no human uPA. Mouse uPA bound only to mouse uPAR and not human uPAR and, thus, could not interact with human uPAR-expressing HEK-293 cells in xenografts. Nevertheless, both mouse and human uPAR significantly increased HEK-293 cell metastasis into the lungs. The activity of human uPAR suggests that uPAR may promote cancer metastasis independent of uPA. Candidate mechanisms include its effects on adhesion, migration, and Rac1 activation.

FOCUS on FOCIS: Combined chemo-immunotherapy for the treatment of hormone-refractory metastatic prostate cancer

Immunotherapy has emerged as another treatment modality in cancer. The goal of immunotherapy in advanced cancer patients does not have to be the complete eradication of tumor cells but rather the restoration of a dynamic balance between tumor cells and the immune response. Appropriate combination of tumor mass reduction (by surgery and/or chemotherapy) and neutralization of tumor-induced immunosuppression might set the right conditions for the induction of anti-tumor immune response by active immunotherapy. We review experimental basis and key concepts of combined chemo-immunotherapy and document its principles in the case report of patient with hormone refractory metastatic prostate cancer with sinister prognosis. More than four hundred prostate cancer patients have been treated with DC-based immunotherapy and tumor-specific immune responses have been reported in two-thirds of them. In half of these patients, DC immunotherapy resulted in transient clinical responses. Tregs, among other factors, potently inhibit tumor-specific T cells. Prostate cancer patients have elevated numbers of circulating and tumor infiltrating Tregs and there is evidence that Tregs increase tumor growth in vivo. Because of the high frequency of circulating Tregs in our patients, we first administered metronomic cyclophosphamide. After obtaining IRB approval, we started regular vaccinations with dendritic cells (DCs) loaded with killed prostate cancer cells. In accordance with the principles of combined immunotherapy, we continued palliative chemotherapy with docetaxel to reduce the tumor cell burden. DC-based vaccination induced prostate cancer cell-specific immune response. Combined chemo-immunotherapy consisting of alternate courses of chemotherapy and vaccination with mature DCs pulsed with LNCap prostate cancer cell line led to the marked improvement in the clinical and laboratory presentation and to the decrease of PSA levels by more than 90%.

Effects of bortezomib on bone disease in multiple myeloma

Bone disease is a well-recognized feature of multiple myeloma (MM), with skeletal-related complications (including pathologic fractures, spinal cord compression, hypercalcemia, and pain) affecting greater than 80% of patients. For many patients, a pathologic fracture or severe bone pain resulting from bone destruction within the marrow cavity is the sentinel event heralding a diagnosis of MM [1]. Importantly, bone disease in MM differs from that of other malignancies. Usually, osteoclast-mediated bone resorption is closely followed both spatially and temporally by osteoblastmediated bone formation. In MM, however, this coupling does not occur even following years of complete remission. Rather bone disease in MM results both from the increased proliferation and activation of osteoclasts and the suppression or absence of osteoblast activity [2]. This finding explains the clinical observation that bone scans (which detect bone formation) in approximately half of patients with MM are normal despite the presence of severe osteolytic disease. Intravenous bisphosphonates (pamidronate and zoledronic acid) bind to bone at sites of exposed hydroxyapatite to limit osteoclast-mediated bone resorption and are the current primary therapy for MM bone disease [3]. Despite bisphosphonate therapy, however, 50% of patients experience a skeletal-related event at the time of relapse [4]. Importantly, however, bisphosphonates target only mature osteoclasts and do not appear to affect osteoblast activity [5]. Further, bisphosphonate therapy is not without risk as MM patients have the highest incidence (among all groups of patients with malignancies receiving bisphosphonate therapy) of avascular osteonecrosis of the jaw [6]. Given these limitations, new strategies to improve skeletal-related outcomes are warranted. In this issue of the American Journal of Hematology, Pennisi et al. [7] demonstrate a skeletal anabolic effect for bortezomib, a first in class proteasome inhibitor with antineoplastic activity in both newly diagnosed and relapsed MM. The authors report that bortezomib (but not melphalan) treatment of immunodeficient mice engrafted with human myeloma cells increased bone formation in previously implanted nonfetal rabbit bone, as documented by both radiographic and bone histomorphometric analyses. The increase in bone formation was associated with an increase in osteoblast numbers along with a simultaneous reduction in the number of osteoclasts. Importantly, however, significant heterogeneity in both the skeletal anabolic and MM cell responses to proteasome inhibition was found. Thus, increases in bone mass were not uniformly found in all subjects treated with bortezomib, and changes in bone mass did not appreciably correlate with the changes in MM tumor cell burden (i.e., subjects with a significant anti-MM cell response to bortezomib may have had little or no bone anabolic response). Accordingly, though the data presented are interesting, it is important to place them in the context of our current understanding of MM bone disease. Over the past several years, the importance of the bone marrow microenvironment in the establishment and progression of MM and MM bone disease has received increasing attention. In this context, the report by Pennisi et al. follows a growing body of literature examining the role of bortezomib in modulating the activity of primary bone cells, namely osteoblasts and osteoclasts. Although the mechanism(s) by which proteasome inhibition leads to increased osteoblast activity has not been firmly established, previous work suggests that proteasome inhibition decreases proteolytic degradation of the Gli3 protein, leading to increased bone morphogenetic protein-2 (BMP-2) expression in osteoblastic cells [8]. Other studies have shown that proteasome inhibition can also increase the activity of the osteoblast transcription factor Runt-related transcription factor 2 (Runx2)/core binding factor a1 (Cbfa1) [9]. These studies are consistent with a recent report by Mukherjee et al. which demonstrated that in mice, bortezomib induces the Runx2/Cbfa1-dependent differentiation of mesenchymal stem/progenitor cells into osteoblasts, leading to new bone formation [10]. In addition, bortezomib also appears to decrease levels of the Wnt pathway inhibitor DKK-1 in patients with MM [11]. As demonstrated in preclinical mouse models of MM, increases in Wnt signaling (and hence osteogenic differentiation) lead to decreased MM bone disease and tumor growth within the bone marrow microenvironment [12,13]. In total, these results are consistent with the increases in bone formation markers demonstrated to occur in MM patients treated with bortezomib [14,15]. As further shown by Pennisi et al., bortezomib also appears to influence osteoclast activity. Although the molecular mechanism(s) by which bortezomib inhibition of proteasome activity leads to decreased osteoclast activity is somewhat unclear, inhibition of nuclear factor-kappa B (NFjB) signaling (perhaps through decreases in TNF Receptor Associated Factor 6 (TRAF6) levels [16] or stabilization of inhibitor of NF-jB (IjB) levels [17]) appears to be important. In turn, NF-jB inhibition serves to limit both the ability of osteoclast precursors to differentiate into mature osteoclasts and the capacity of mature osteoclasts to resorb bone [18]. Importantly, Terpos et al. have shown that bortezomib can reduce serum levels of receptor activator of NF-

Major Shrinking of Residual Tumor Cell Burden and Achievement of Molecular Remissions in Myeloma Patients Undergoing Post-Trasplant Consolidation with Bortezomib, Thalidomide and Dexamethasone: A Qualitative and Quantitative PCR Study

Background and aims. In multiple myeloma (MM) molecular remission (MR) is usually not achieved with autologous transplantation (ASCT) as opposed to allogeneic transplantation where it occurs frequently and associates to an improved outcome. Aim of this study was to assess, by qualitative and quantitative PCR, the impact of a consolidation treatment, including Bortezomib/Thalidomide/Dexamethasone (VTD) on residual MM cells in patients achieving a good clinical response after ASCT.Patients and methods: Inclusion criteria were:a documented complete or very good partial remission (CR or VGPR) following ASCT delivered as first line treatment;no previous treatment with thalidomide and bortezomib;presence of a molecular marker based on the immunoglobulin heavy chain rearrangement (IgH-R).VTD had to be started within 6 months from ASCT. Each cycle consisted of:Bortezomib 1.6 mg/m2 as an IV injection once weekly (on days 1, 8, 15, 22) followed by a 13-day rest period (days 23–35);Thalidomide at the initial dose of 50 mg/day PO once daily, with increments of 50 mg every 7 days up to 200 mg;Dexamethasone 20 mg/day PO once daily, on days 1 to 4, 8 to 11 and 15 to 18 followed by a 17-day rest period (days 19–35).A total of 4 cycles were delivered. MRD was assessed on bone marrow (BM) samples at diagnosis, study entry, after two VTD courses, at the end of treatment and then at six months intervals. Qualitative and quantitative PCR analysis were carried out using IgH-R-derived patient-specific primers as already described (Voena et al, Leukemia 1997; Ladetto et al, Biol Bone Marrow Transpl 2000).Results: Forty pts were enrolled and are evaluable at study entry. 20% of patients did not receive the whole planned treatment, but were nevertheless included in MRD analysis. Median follow-up from study entry is currently 21 months. Qualitative PCR has been performed on the whole population (overall 198 samples). Six patients converted to MR (defined as two consecutive PCR-negative samples, spaced at least three months) while two patients had an isolated PCR-negative result. Patients in MR persisted in their status with the exception of one molecular relapse at 24 months. No clinical relapse has been so far observed in MR patients at a median follow-up of 26 months (18–30). Among patients not achieving MR we observed eight relapses occurring at a median time of 12 months (4–26). Quantitative PCR has been performed on 20 patients (overall 105 samples). Median tumor bulk at diagnosis was 157000 (35-925000) IgH-R/106 diploid genomes (dg). It shrunk to 440 (3-420000) following ASCT and to 17 (0-113000) following VTD. The effect of VTD was detectable both in patients in VGPR and CR. Follow-up analysis by real time PCR at six, 12 and 18 months in persistently PCR-positive patients revealed stable MRD levels in 66% of patients and a growth greater than one log in 33%. Notably, patients who already relapsed were characterized by a tumor load persistently greater than 100 IgH-R/106dg except one who showed a transient tumor cell reduction after 2 VTD courses followed by a sharp increase in his MRD level.Conclusions: Post-transplant VTD consolidation is active on residual plasma cells surviving ASCT. Moreover a proportion of CR/VGPR MM enters MR which might persist up to 30 months. Thus, new non-chemotherapeutic agents can substantially improve the quality of remission in MM patients even in case of optimal response to ASCT.