Search

11027 Background: Some recent publications indicated that the use of G-CSF could be connected to an increase in CTC as well as elevated levels of tumor markers such as CA 27.29. In the SUCCESS Trial CTC and CA27.29 are examined before and after adjuvant chemotherapy (CHT) in 3754 breast cancer patients (pts). Methods: The SUCCESS Trial is a phase III trial comparing FEC-Docetaxel vs. FEC-Doc-Gemcitabine regime and 2 vs. 5 years of treatment with zoledronate in patients with primary breast cancer (BC) (N+ or high risk). Blood samples are taken before and after CHT. CTC were assessed with the CellSearchSystem (Veridex, Warren, USA). After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labeled with anti-cytokeratin (8,18,19) and anti-CD45 antibodies to distinguish epithelial cells and leukocytes. CA27.29 has been measured with ST AIA-PACK Ca27.29 reagent using MUC-1 for AIA-600II (Tosoh Bioscience, Tessenderlo, Belgium). The cutoff for CA27.29 is 32 U/ml and >1 cell for the CTC analysis. Patients were grouped to CTC/CA27.29 raise or no raise and 1 to 6 cycles with G-CSF or no G-CSF at all. Results: Data on 1510 pts are available for CTC analysis. 745 pts (49%) received at least one course of G-CSF. 117 pts (8%) showed an increase in CTC after CHT. In this group 52 (3%) pts received G-CSF and 65 (4%) did not. 693 pts with stable or decreased CTC received G-CSF (46%) and 700 did not (46%). There was no significant difference (p=0.29). The analysis of CA27.29 is based on the data of 2556 pts. 1252 pts (49%) received at least one course of G-CSF. 338 pts (13%) exceeded the threshold for CA27.29 only after CHT. In this group 209 pts (8%) received G-CSF and 129 (5%) did not. 1043 pts with stable or decreased CA27.29 received G-CSF (41%) and 1175 did not (46%). This difference was highly significant (p [Table: see text]

Abstract #6109 Background: The non-steroidal aromatase inhibitor (AI) anastrozole is superior to tamoxifen in terms of efficacy and safety in the adjuvant treatment of postmenopausal patients with hormone receptor-positive (HR+) early breast cancer (EBC). The aim of the current study was to evaluate the cost-effectiveness of anastrozole versus tamoxifen from the German health care perspective. Methods: A probabilistic Markov model was developed using data from the published literature and expert opinion to project trial outcomes to 25 years based on a cohort of 1000 postmenopausal women with HR+ EBC. Resource use data and costs were obtained from standard sources and expert opinion. Utility scores for the different heath states were obtained from a patient-based utility study using the standard gamble technique. Results were expressed as incremental cost-effectiveness ratios (ICERs) per quality adjusted life-years (QALYs) gained. Costs and benefits were discounted at 5% annually. Results: Total costs over the 25 year period for anastrozole and tamoxifen were €12,422 and €6,757, respectively. The cost per life year (LY) for anastrozole compared to tamoxifen was €24,822/LY gained. The ICER of anastrozole compared to tamoxifen was €21,050/QALY gained (95% CI €9,841 - €62,344). The result remained robust to all parameters tested in the sensitivity analysis. Cost-effectiveness probability curves indicated a >90% probability that the cost per QALY gained with anastrozole would be Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6109.

Expression of interleukin-6 (IL-6) and IL-6 receptors has been demonstrated in Hodgkin and Reed-Sternberg (H and RS) cells in vitro and in vivo. In order to evaluate the clinical significance of IL-6 serum levels in patients with Hodgkin's disease (HD), we tested the sera of 56 untreated patients with HD by means of a sensitive sandwich ELISA. While IL-6 was only rarely detectable in healthy controls or patients with non-Hodgkin's lymphoma, 32 of 56 patients (57 per cent) had detectable IL-6 levels (range 12-32 pg/ml). The rates of detectable IL-6 levels and the median levels were not correlated with age, sex, histological subtype, stage or the presence of B-symptoms, nor with any of a wide spectrum of laboratory parameters tested, including erythrocyte sedimentation rate, total leukocyte and lymphocyte counts, serum levels of soluble CD8, CD25 or CD30. The rates of complete remissions and freedom from treatment failure were not different in IL-6-negative and IL-6-positive patients. Except in one of 23 follow-up sera taken after therapy, IL-6 was no longer detectable even for patients who suffered from progressing disease, suggesting that the neoplastic H and RS cells are not the major source of circulating IL-6.

1054 Background: Breast cancer patients are increasingly exposed to anthracyclines and/or taxanes during perioperative therapy. Reinduction with these agents for treatment of metastatic disease is frequently limited by drug resistance or cumulative toxicity. Consequently, there is a need to develop new regimens free of anthracyclines or taxanes. Methods: This randomised phase II trial investigated pretreated MBC patients who were attributed to one of three gemcitabine-based regimens applied at 3-week intervals. Patients received either Gem/Vin (gemcitabine 1,000 mg/m2 days 1+8 plus vinorelbine 25 mg/m2 days 1+8) or Gem/Cis (gemcitabine 1,000mg/m2 days 1+8 plus cisplatin 30 mg/m2 days 1+8) or Gem/Cap (gemcitabine 1,000 mg/m2 days 1+8 plus capecitabine 650 mg/m2 bid orally days 1+14). ORR was evaluated as a primary end-point, while TTP, OS, and safety were analysed as secondary endpoints. Results: 134 eligible MBC patients were randomized and received either Gem/Vin (n=41), Gem/Cis (n=44), or Gem/Cap (n=49)...

The Hodgkin's lymphoma-derived cell line L540, which exhibits multiple marker chromosomes and a genotype characteristic of T-cell origin, showed expression of MET oncogene. L540 was studied by in situ hybridization for chromosomal rearrangements concerning the regions where genes for TCRA and TCRB chains, for MET oncogene, and for rRNA are located. All four genes were demonstrated to be involved in the formation of marker chromosomes. Especially remarkable was the participation of rDNA-bearing regions in two different translocations.

Hodgkin and Sternberg Reed (H and SR) cells, the putative malignant cells of Hodgkin's disease carry regularly T-cell activation antigens, like CD30 and CD25 (low affinity IL-2 receptor). We have investigated the Hodgkin cell line L540, bearing characteristic markers of H and SR cells for its expression of the low affinity IL-2 receptor (IL-2R) and for IL-2. Expression of the low affinity IL-2R was found on mRNA level, by detection of specific 3.5 kb and 1.4 kb mRNA and on the protein level by immunoprecipitation of a 55,000 mol. wt molecule from detergent extracts of surface iodinated cells, however IL-2 specific mRNA was not detected. Scatchard plot analysis revealed the presence of 2 × 10 3 high affinity IL-2Rs. Crosslinking experiments directly demonstrated the high affinity IL-2R to consist of the 55,000 mol.wt light chain (L), and the 70/75,000 (H1/H2) heavy chains. IL-2 was rapidly internalized by these receptors, suggesting that they can be functional. The expression of functional IL-2Rs might be involved in induction or differentiation of Hodgkin's disease.

Rearrangements of immunoglobulin and T cell receptor (TCR) genes have been demonstrated in malignant lymphoid tumors of B and T cell origin. In Philadelphia chromosome-positive chronic myeloid leukemia and acute lymphocytic leukemia cells the bcr and c-abl genes are reorganized and transcripts composed of both genes are expressed. We analyzed the organization of bcr, immunoglobulin and TCR genes in malignant lymphomas. Our data show that in all B cell lymphomas analyzed the JH genes and in some cases also the J kappa genes were rearranged. In a Burkitt lymphoma and in a Kil lymphoma distinct rearranged TCR gamma fragments were detected, in a second Burkitt lymphoma two rearranged TCR beta gene fragments occurred together with a rearranged JH gene fragment. In two T cell lymphomas rearranged TCR beta genes were observed; one of these lymphomas also carried rearranged TCR gamma and JH genes. In Hodgkin's disease in 3 out of 7 cases rearranged immunoglobulin genes were detected. In 1 case, which was diagnosed as a follicular hyperplasia, rearranged JH and TCR gamma fragments appeared. In none of the analyzed lymphomas could bcr rearrangements be observed.

11005 Background: Several trials have shown an earlier detection of metast. disease in breast cancer pts by the use of TU- markers. Whether this lead time advantage can be translated into improved outcome is discussed controversially. We prospectively evaluated the role of Ca27.29 in breast cancer pts at primary diagnosis and during adjuvant systemic treatment within the German SUCCESS-Trial (n=3658 pts planned). Methods: The SUCCESS-Trial compares sequential chemotherapy with FEC-Docetaxel(Doc) vs. FEC- DocGemcitabine, followed by zoledronate 2 vs. 5 years in primary high risk N0 and N+ breast cancer pts. Ca27.29 levels were assessed using the specific ST AIA-PACK Ca27.29 reagents directed against MUC-1 for AIA-600II (Tosoh Bioscience, Tessenderlo, Belgium). Cut-off levels for the assay was 24 U/ml. Results: We analyzed 1098 breast cancer pts prospectively before start and after completion of chemotherapy. 21% of pts (n=226; median 17U/ml; range 4–410) had tumor marker elevation = 24 U/ml before and 48% (n=524; median 23U/ml; range 4–198) after completion of chemotherapy. Ca27.29 levels before and after chemo correl. significantly (p No significant financial relationships to disclose.

CT-associated cognitive dysfunction, which frequently manifests as subtle changes in memory, concentration, and ability to remain focused or organized, can adversely affect cancer pts’ QOL. EPOLYM, a prospective, international, multicenter, open-label, phase IIIb trial has been conducted to investigate the efficacy, safety, and clinical outcomes, including effect on cognitive function, of epoetin alfa 40,000 IU sc QW in anemic (Hb

Three Hodgkin's disease-derived cell lines were analysed for the organization of immunoglobulin and T cell receptor genes, for the expression of the interleukin 2 (IL-2) receptor gene, and for the cellular oncogene c-myc. All three cell lines have characteristic genotypic markers of lymphoid cells and can be classified as immature lymphoid cells with respect to the incomplete expression of their antigen receptor genes. This immature genotype is in contrast to the expression of activation antigens Ki-1 (CD 30), IL-2 receptor (CD 25), and HLA-DR. The results obtained are in agreement with studies obtained from primary Hodgkin's lymphomas, which are activated by as yet unknown mechanisms.

Three Hodgkin-derived cell lines (L428, L540, and CO) were studied for rearrangements and expression of immunoglobulin and T-cell receptor genes, and their genotype was compared to the phenotype. As far as the genotype is concerned, all 3 cell lines have characteristics of lymphoid cells; L428 of B, and L540 and CO of T-cell origin. L428 cells have one Ig heavy chain allele rearranged to C gamma and transcribed into RNA, while the second is deleted. Furthermore, L428 cells show an unusual immunoglobulin kappa light chain gene rearrangement involving deletion of the kappa constant gene in one allele, while the remaining kappa and lambda loci are in germline configuration. L540 and CO have, in contrast to L428 cells, the immunoglobulin genes in germline and T-cell receptor genes rearranged. The T-cell receptor beta and gamma genes are rearranged in both L540 and CO, whereas a rearrangement in the alpha locus was detected in L540 cells only. RNA of the size of functional beta chain transcripts was found in CO cells and of the size of functional alpha chain transcripts in L540 cells. All 3 cell lines are classified as immature lymphoid cells with respect to the limited expression of B- and T-cell antigens, respectively, and to the incomplete expression of their antigen receptor. The immaturity of lymphoid differentiation contrasts with the expression of activation antigens, i.e. Ki-1, Ki-24, HLA-DR, and IL-2 receptor. The immaturity of the cells excludes the possibility that the cells were activated along the physiological pathway, i.e. by interaction of the cell with antigen. The results obtained on the cell lines are in accordance with in vivo studies and suggest that Hodgkin and Sternberg-Reed cells are immature lymphoid cells which are activated by a still unknown mechanism.

A method is described for the efficient purification of human B lymphocytes from peripheral blood by magnetic separation. Biotinylated, superparamagnetic particles were coupled to target cells by fluorescein isothiocyanate conjugated avidin and biotinylated monoclonal antibodies directed against cell surface antigens. This combination permitted flow cytometric control of the magnetic separation. Ficoll-Paque-separated peripheral blood mononuclear cells were first eliminated from monocytes by leucine-methyl ester treatment. B cells were enriched to 97% after magnetic depletion of CD3-positive T cells and magnetic enrichment of CD20-positive B cells. The separated B cells could be induced to proliferation and antibody production by various in vitro stimuli.

In a case of CML with a variant Philadelphia translocation (Ph1 or Ph) t (22;22) (q11;q13) in bone marrow cells and unstimulated peripheral blood cells, no cytogenetically detectable involvement of chromosome 9 was observed. Southern blot experiments using probes specific for bcr and c-sis however revealed rearrangement of the bcr, but not of PDGFB (c-sis) gene. Northern blot analysis of bone marrow RNA showed a very weak signal with the c-sis probe, while in a lymph-node biopsy PDGFB m-RNA could not be detected. Chromosomal in situ hybridization gave evidence for translocation of c-abl from chromosome 9 to Ph and of PDGFB from chromosome 22 to chromosome 9, as the result of a threefold translocation t(9;22;22).

Cellular oncogenes are frequently activated or deregulated in human malignant tumors. We have analyzed the expression of cellular oncogenes in human leukemia by Northern blot experiments in a case of acute myeloid leukemia (AML). In this case the cellular oncogenes N-myc, c-myb, c-fes and c-met were expressed at high levels. This is in contrast to normal peripheral blood mononuclear cells (PBMC) or chronic lymphocytic leukemia (CLL) cells, where these genes could not be detected. c-src2-specific RNA was not seen in either AML or PBMC cells but readily appeared in CLL cells. This appears to represent further evidence that in leukemic cells multiple cellular oncogenes might be activated.

In the present study we use two monoclonal anti-idiotope antibodies (Ac38 and Ac146; Ab1) against the germ line-encoded, lambda 1 chain-bearing and (4-hydroxy-3-nitrophenyl)acetyl (NP)-binding antibody B1-8 (Ab1) for the induction of complementary antibodies (Ab3) and ask the question to what extent antibodies bearing B1-8-like idiotopes (Ab3 beta) are represented in the Ab3 population. In this experimental system, Ab3 beta is distinguished from the remaining Ab3 population (Ab3 alpha) by three properties which Ab3 beta may share with B1-8: (a) the binding of NP, (b) the binding to Ac146 (if induced by Ac38) or the binding to Ac38 (if induced by 146) and (c) that they carry lambda 1 chains. Antibodies with all three properties are induced in low amounts by both anti-idiotopes. Also, Ac146 induces only Ab3 alpha (bearing kappa chains and not binding NP and Ac38). In contrast, Ac38 triggers almost exclusively a lambda 1 chain-bearing response, i.e. Ab3 beta. The response has an unusually large size, reaches its maximum after a week and is long-lasting. An analysis at the level of lipopolysaccharide-reactive precursor B cells demonstrates that, in this case, cells expressing Ab3 beta occur at exceedingly high frequency (approximately equal to 10(-3] and are at least 10 times more frequent than cells expressing Ab3 alpha. The high frequency of Ab3 beta-expressing cells correlates with the contribution of several VH, D and J genes to the expression of this particular idiotope. In the case of the Ac146 anti-idiotope antibody, the response is dominated by Ab3 alpha. Ab3 beta represents less than 10% of the total response, reaches maximal levels 2 weeks after immunization and declines rapidly. This correlates with a low frequency of precursor B cells expressing Ab3 beta (approximately equal to 10(-5] and a restriction of the corresponding idiotope to rare VH-D combinations, caused presumably by a stringent contribution of the H chain to this idiotope which covers the NP-binding site. Our data suggest that anti-idiotypic antibodies (Ab2) against Ab1 will preferentially induce antibodies idiotypically related to Ab1 if the corresponding idiotopes are expressed in high frequency in the B cell compartment. This is expected in cases where Ab2 recognizes an idiotope that can be formed by many germ line-encoded VH-D-VL-combinations.

Background In elderly patients with HER2-positive breast cancer, few data on efficacy and toxicity of adjuvant trastuzumab treatment exists since older patients were in general excluded from large randomized studies. This prospective observational study aimed to confirm the beneficial findings from pivotal trials in age cohorts ≥65years. Materials and Methods There were no restrictions for recruitment with respect to age or concomitant/sequential adjuvant medication. Long-term relapse/survival status of the patients was assessed once a year. Results Among the 3940 evaluable patients enrolled between 2006 and 2012 at 339 institutions, 507 were aged between 65 and 69years, with another 507 patients ≥70years. Elderly patients suffered from significantly more advanced primary tumors. Preceding or concomitant chemotherapy showed decreasing aggressiveness with patient's age. Trastuzumab treatment was stopped prematurely in only 11% of the elderly, but more often than in younger patients ( p =0.0008). With 453 events hitherto reported, elderly patients did not exhibit an inferior relapse-free survival when adjusted for other relevant prognostic factors (hazard ratio: 1.01 per year; p =0.24). Three-year overall survival was significantly lower in the population older than 64years than in younger patients (94.2% vs. 96.8%, p =0.0011). Conclusions To our knowledge, our population of elderly patients treated with adjuvant trastuzumab is the largest analyzed so far. The beneficial long-term results were comparable to those in the younger cohorts. Although the risk of cardiotoxicity increased significantly with age, it also remained manageable in older patients. Thus, chronological age alone should not preclude HER2 antibody treatment.

In the present analysis we dissect the idiotype repertoire, independently of hapten-binding specificity, by immunizing different strains of mice with cross-linked monoclonal anti-idiotope antibodies against antibody B1-8. B1-8 is a monoclonal antibody with specificity for the hapten (4-hydroxy-3-nitro-phenyl)acetyl (NP) and carries a germ line gene-encoded variable region. The results demonstrate that the expression of B1-8 idiotopes and their association with each other and with NP-binding specificity are strain-specific. Certain idiotopes are expressed on antibodies differing in antigen-binding specificity, whereas one of the idiotopes appears strictly associated with NP-binding antibodies. The genetic analysis provides strong evidence that the strain specificity of the idiotope repertoire is a result of V region polymorphism in the mouse.

IgG subclass levels to Pseudomonas aeruginosa alginate, alkaline proteinase, elastase and exotoxin A in sera of healthy adults, non-infected and infected cystic fibrosis patients were investigated by enzyme linked immunosorbent assay. Whereas healthy adults and non-infected cystic fibrosis patients revealed mostly negative IgG subclass levels to the four antigens, infected cystic fibrosis patients had significantly elevated IgG1, IgG2, IgG3 and IgG4 levels to both the protein antigens as well as the polysaccharide antigen. The study does not support previous findings of an impaired natural IgG2 response to polysaccharide antigen. The study does not support previous findings of an impaired natural IgG2 response to polysaccharide in chronically infected cystic fibrosis patients.

When the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) is presented on different carrier molecules, different anti-NP antibody responses are stimulated. On stimulation with NP-lipopolysaccharide (LPS) [T-independent type 1 (TI-1) antigen] kappa + antibodies are the major population, whereas on stimulation with NP-Ficoll [T-independent type 2 (TI-2) antigen], NP-keyhole limpet hemocyanin (KLH) or NP-chicken gamma globulin (CG) [T-dependent (TD) antigens], lambda 1+ antibodies dominate. The relative contribution of idiotopes Ac38 or Ac146 to the lambda 1+ anti-NP response was also different on comparison of TI-1 with TI-2 or TD anti-NP responses. We investigated whether light chain- or idiotype-specific T cells are responsible for these differences. Analysis of the anti-NP response of nude mice after immunization with NP-Ficoll showed lambda 1 dominance. Likewise primary adoptive transfer experiments using carrier-specific T cell lines to reconstitute the TD anti-NP response to NP-KLH or NP-CG, showed that help from carrier-specific T cells alone is capable of stimulating the characteristic lambda 1 dominant response. No significant difference could be found in the levels of Ac38 and Ac146 idiotope expression between mice reconstituted with splenic T cells and those reconstituted with T cell lines. These results suggest that light chain- or idiotype-specific T cells are required neither for the production of lambda 1 light chain dominance, nor for the appearance of idiotopes characteristic of the primary anti-NP response. The possible reasons for differences seen in both light chain and idiotope expression between primary anti-NP responses to the TI-1 antigen NP-LPS and those to TD or TI-2 antigens are discussed.

The thymus is an important site of sensitization of autoreactive B and T lymphocytes in myasthenia gravis (MG). We have investigated clonal diversity of B or T cells in the thymus of patients with MG by Southern blot experiments using probes specific for immunoglobulin heavy (IgH) and light chain (IgL) genes and for T cell receptor (TCR) beta- and gamma-chain genes. This method allows to detect individual clones of B or T cells if they represent at least 1% of the total cell population. We investigated thymus glands from 14 patients who underwent thymectomy. Single rearranged fragments could be demonstrated with a TCR gamma-specific probe in DNAs from both normal donors and MG patients. TCR beta gene rearrangements occurred mainly in the C beta 1 region. However, single rearranged bands could not be detected with either TCR beta, JH or with J kappa specific probes. Thus any single autoimmune B and T cell clone present in the myasthenic thymus represents presumably less than 1% of all thymocytes.

Epstein-Barr virus-(EBV-)transformed B cells were obtained from a patient with myasthenia gravis, who was also the donor of a T cell line specific for acetylcholine receptor (AChR). After three months of culture, the EBV-transformed B cells could effectively present native membrane-bound AChR to autologous AChR-specific T cells.

In the response to NP-lipopolysaccharide or NP-Ficoll predominantly anti-NP antibodies of the IgM class are produced in mice with lower amounts of IgG3 and IgG2b but little or no IgG1 and IgG2a. In contrast, in the primary T-dependent response to NP-keyhole limpet hemocyanin (KLH) or NP-chicken gamma globulin high amounts of all IgG isotypes are induced. To investigate whether isotype-specific T cells are responsible for these differences we carried out cell transfer experiments using carrier-specific T cell lines. Two such lines were established and one of the two could be cloned. Upon activation by antigen the T cell lines induced unprimed syngeneic splenic B cells to proliferate and differentiate into antibody-secreting cells in vitro in an antigen-nonspecific way. Antigen-specific activation of unprimed B cells in a cell transfer system in vivo showed that high concentrations of hapten-specific antibodies of all IgG isotypes are induced through both carrier-specific T helper lines. The isotypic pattern of these antibodies is similar to that produced via heterogeneous splenic T cells in the cell transfer system, or in normal animals on immunization with the same antigen. These results suggest that isotype-specific T cells are not required for the production of IgG isotypes in a primary anti-NP response and thus not responsible for the differences seen in isotypic patterns between T-dependent and T-independent responses.

510 Background: We have recently described tumor-infiltrating lymphocytes (TILs) as predictors of pathological complete response (pCR = ypT0ypN0) to neoadjuvant carboplatin-based chemotherapy in th...