Drug response prediction in high-risk multiple myeloma.

A Drug Response Prediction (DRP) score was developed based on gene expression profiling (GEP) from cell lines and tumor samples. Twenty percent of high-risk patients by GEP70 treated in Total Therapy 2 and 3A have a progression-free survival (PFS) of more than 10years. We used available GEP data from high-risk patients by GEP70 at diagnosis from Total Therapy 2 and 3A to predict the response by the DRP score of drugs used in the treatment of myeloma patients. The DRP score stratified patients further. High-risk myeloma with a predicted sensitivity to melphalan by the DRP score had a prolonged PFS, HR=2.4 (1.2-4.9, P=0.014) and those with predicted sensitivity to bortezomib had a HR 5.7 (1.2-27, P=0.027). In case of predicted sensitivity to bortezomib, a better response to treatment was found (P=0.022). This method may provide us with a tool for identifying candidates for effective personalized medicine and spare potential non-responders from suffering toxicity.

Gene expression risk signatures maintain prognostic power in multiple myeloma despite microarray probe set translation.

INTRODUCTION: Gene expression profiling (GEP) risk models in multiple myeloma are based on 3'-end microarrays. We hypothesized that GEP risk signatures could retain prognostic power despite being translated and applied to whole-transcript microarray data. METHODS: We studied CD138-positive bone marrow plasma cells in a prospective cohort of 59 samples from newly diagnosed patients eligible for high-dose therapy (HDT) and 67 samples from previous HDT patients with progressive disease. We used Affymetrix Human Gene 1.1 ST microarrays for GEP. Nine GEP risk signatures were translated by probe set match and applied to our data in multivariate Cox regression analysis for progression-free survival and overall survival in combination with clinical, cytogenetic and biochemical risk markers, including the International Staging System (ISS). RESULTS: Median follow-up was 66 months (range 42-87). Various translated GEP risk signatures or combinations hereof were significantly correlated with survival: among newly diagnosed patients mainly in combination with cytogenetic high-risk markers and among relapsed patients mainly in combination with ISS stage III. CONCLUSION: Translated GEP risk signatures maintain significant prognostic power in HDT myeloma patients. We suggest probe set matching for GEP risk signature translation as part of the efforts towards a microarray-independent GEP risk standard. (ClicinalTrials.gov identifier: NCT00639054).

Increased level of both CD4+FOXP3+ regulatory T cells and CD14+HLA-DR⁻/low myeloid-derived suppressor cells and decreased level of dendritic cells in patients with multiple myeloma.

Patients with multiple myeloma (MM) suffer from a general impaired immunity comprising deficiencies in humoral responses, T-cell responses as well as dendritic cell (DC) function. Thus, to achieve control of tumour growth through immune therapy constitutes a challenge. Careful evaluation of the immune status in patients with MM seems crucial prior to active immune therapy. We evaluated the proportion of both, DC, Treg cells and myeloid-derived suppressor cells (MDSC) in peripheral blood from patients with MM at diagnosis and in remission as well as patients with monoclonal gammopathy of undetermined significance (MGUS). We found that the proportion of both myeloid (m) DC and plasmacytoid (p) DC in patients at diagnosis was lowered compared to control donors, while only the proportion of pDC in patients in remission and with MGUS was significantly lower than in controls. The proportion of CD4+FOXP3+ Treg cells was increased in patients at diagnosis and not in patients in remission or with MGUS. Also, Treg cells from patients with MM were functionally intact as they were able to inhibit proliferation of both CD4 and CD8 T cells. Finally, we observed an increase in the proportion of CD14+HLA-DR⁻/low MDSC in patients with MM at diagnosis, illustrating that this cell fraction is also distorted in patients with MM. Taken together, our results illustrate that, both mDC, pDC, Treg cells and MDSC are affected in patients with MM underlining the fact that the immune system is dysregulated as a consequence of the disease.

Early infections in patients undergoing high-dose treatment with stem cell support: a comparison of patients with non-Hodgkin lymphoma and multiple myeloma.

BACKGROUND: Infections are life-threatening complications in patients undergoing high-dose chemotherapy with stem cell support (HDT). Knowledge of the infectious pathogens is essential to make a safe outpatient setting. METHODS: We conducted a retrospective study of 208 patients treated with HDT. The population included non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) patients. No patients received prophylactic antibacterial treatment. RESULTS: Pathogens were isolated from 44% of all patients. MM patients more frequently had multiple pathogens in blood cultures (38% versus 25%). Transplantation related mortality was similar between the groups. CONCLUSION: The frequency of isolated pathogens, positive blood cultures, and the diversity of pathogens were higher in MM patients as compared to NHL patients. However, this did not translate into higher transplantation-related mortality, probably because broad-spectrum antibiotic treatment could be initiated immediately. A safe outpatient setting with prophylactic antibiotic treatment is dependent on continuous collection and registration of microbiological findings.

The polymorphism IL-1beta T-31C is associated with a longer overall survival in patients with multiple myeloma undergoing auto-SCT.

Proinflammatory cytokines are suspected to play a role in the pathogenesis of multiple myeloma (MM). Therefore, it is possible that inborn genetic variations leading to a modified expression of these cytokines will influence the outcome for these patients. We investigated 348 MM patients undergoing high-dose melphalan treatment followed by Auto-SCT and examined the influence of single nucleotide polymorphisms (SNPs) in genes involved in the inflammatory response. We found that the polymorphism IL-1beta T-31C significantly influenced overall survival (OS; P=0.02) and that carriers of the variant C-allele had a significantly longer survival than homozygous wild-type allele TT-carriers (relative risk 0.6 (95% CI=0.5-0.9); P=0.008). The polymorphisms IL-6 G-174C, IL-10 C592A, PPARgamma2 Pro(12)Ala, COX-2 A-1195G, COX-2 T8473C and NFKB1 ins/del did not influence the OS in this group of patients. Furthermore, homozygous carriers of the variant allele of IL-1beta T-31C were at 1.37-fold (CI=1.05-1.80) increased risk of MM as compared with population-based controls (P=0.02). Our results indicate that IL-1beta is involved in the pathogenesis of MM.

Fucosyl-GM1 in small-cell lung cancer. A comparison with the tumour marker neuron-specific enolase.

BACKGROUND: Recently, the ganglioside Fucosyl-GM1 (FucGM1) has been described as a possible new tumour marker for small-cell lung cancer (SCLC). FucGM1 has been detected in 75% to 90% of SCLC tumours by immunohistochemical analysis and in about 50% of sera from SCLC patients. Neuron-specific enolase (NSE) is a glycolytic enzyme which is expressed in the majority of SCLC tumours and patient sera. PATIENTS AND METHODS: Sera from 156 patients with SCLC were analyzed for FucGM1 with a scintillation proximity assay (SPA), which is a simple and sensitive analysis. Sera were analyzed before the initiation of chemotherapy, and twenty patients were monitored during and after treatment. The concentration of FucGM1 was compared to the tumour marker NSE and related to clinical data and survival. RESULTS: Sixty-three per cent of the patients were positive for FucGM1. The concentrations did not correlate with NSE or clinical data including stage of disease, organ site of metastases or ABO blood group status. Nor did the expression of FucGM1 correlate with survival. As a monitor of clinical response, a correlation was found in 8 out of 20 patients. Eighty-four per cent of the patients were positive for NSE; and 97% were positive for either FucGM1 or NSE. CONCLUSION: We conclude that FucGM1 does not have a clinical role as a tumour marker for patients with SCLC at diagnosis or during treatment.

Serological tumor markers for small cell lung cancer and their therapeutic implications.

Serological tumor markers may become widely used as inexpensive and non-invasive methods of cancer detection. Markers of current interest for small cell lung cancer (SCLC) comprise enzymes, peptides, proteins, and carbohydrates. None of the serological markers for SCLC have yet proven to be of diagnostic value and at present their use is limited to monitoring disease and indicating prognosis. However, whilst serological markers related to the metabolic state of SCLC cells, such as neuron-specific enolase, serum thymidine kinase and tissue polypeptide antigen, may only be used for monitoring patients and for estimating prognosis, the other serological markers under current investigation may be used to indicate new treatment forms. Several novel approaches, including interference in the autocrine growth-regulating loop of SCLC by either peptides or antibodies, have been tried, SCLC is a highly heterogeneous tumor with respect to antigen expression, regulation of growth, and differentiation state. It is therefore important that new interventions are directed against both antigen-positive and antigen-negative tumor cells. For instance, radioisotopes or enzymes coupled to antibodies may be effective by exerting toxicity at some distance from the target. Antigens expressed on SCLC cells, such as peptide receptors involved in growth regulation, carbohydrate antigens like Lewis antigens, carcinoembryonic antigen and the ganglioside fucosylGM1, provide potential targets for antibody-conjugated therapy.

Strain- and age-dependent natural and activated in vitro cytotoxicity in athymic nude mice.

The defect in athymic nude mice with respect to T-lymphocyte number and function is accompanied by increased levels of natural cytotoxicity as well as other immune reactions with potential antitumor effects. With increasing age of immunodeficient mice the take rate of xenotransplanted tumors decreases while the number of cells with T-lymphocyte markers increases and some T-lymphocyte-associated functions become detectable. In the present study the natural cytotoxicity of nonadherent splenocytes from young (4-6 weeks) and aged (about 1 year) BALB/c nu/nu mice was analyzed and compared with that of splenocytes from normal euthymic young and old Balb/c mice on the one hand, and with Bar nu/nu and NCr nu/nu young and old mice on the other. To investigate a possible contribution of T lymphocytes to the cytotoxic effect in athymic mice, LAK cells were generated. For this purpose, the nonadherent splenocytes were exposed in vitro either to IL-2 or to anti-CD3 mAb, specifically to activate T lymphocytes expressing TcR-CD3 in the latter case. Cytotoxic in vitro assays were applied using YAC-1 (NK-sensitive) and P-815 (NK-resistant, LAK-sensitive) target cells in parallel experiments in which splenocytes from young and old donors were used as effector cells. Splenocytes from young immunodeficient mice showed consistently high cytotoxicity with YAC-1 cells. In aged athymic mice, splenocytes stimulated in vitro with anti-CD3 mAb showed cytotoxicity to P-815 (LAK-sensitive) cells. FACS analyses of antigenic markers revealed an increased number of T cells in spleens of aged immunodeficient mice, with differences between mice of the examined strains and a decrease in the number of NK cells in aged mice.

New serum markers for small-cell lung cancer. II. The neural cell adhesion molecule, NCAM.

The neural cell adhesion molecule (NCAM) was recently suggested as a marker for small-cell lung cancer (SCLC). Immunohistochemical analysis demonstrated the presence of the NCAM in 78% of SCLC patients and in 25% of patients with other cancer forms. NCAM was proposed to be the most sensitive marker for SCLC, and it may also be an important prognostic marker for SCLC. We used a competitive ELISA to analyze the concentrations of NCAM in sera from 96 SCLC patients, 16 patients with non-SCLC, 4 patients with other cancer forms, and 16 healthy controls. All sera were collected at the time of diagnosis, before the patients received chemotherapy. The polyclonal antibody used in the assay recognized all three isoforms of NCAM. The concentration of NCAM was related to clinical parameters of the patients such as age, sex, blood group status, stage of disease, organ site involvement of metastases, survival, and expression of the ganglioside fucosyl-GM1 (FucGM1). Sera were considered positive if NCAM concentrations were higher than the mean concentration of healthy controls plus two standard deviations. Twenty-two percent of the sera from SCLC patients were positive for NCAM. No difference in concentration was found between SCLC patients with localized and extensive disease. Serum from one patient with cancer of the thyroid, but no sera from non-SCLC patients or normal healthy controls, was positive. The expression of NCAM did not correlate to any of the clinical parameters, and no correlation was found to the other serum marker, FucGM1.(ABSTRACT TRUNCATED AT 250 WORDS)

New serum markers for small-cell lung cancer. I. The ganglioside fucosyl-GM1.

The ganglioside fucosyl-GM1 (FucGM1) has been suggested as a marker for small-cell lung cancer (SCLC). Immunohistochemical analyses have shown the expression of the ganglioside in tumors in 75 to 90% of patients with SCLC. We have demonstrated that the ganglioside is shedded from SCLC cells both in vitro and in vivo, and that the antigen can be detected in sera from SCLC patients by an immunochemical analysis. The FucGM1 antigen has recently been shown to act as a target for antibody-dependent cellular cytotoxicity. This may provide a rationale for developing immunotherapy against SCLC. We used an immunoassay based on the scintillation proximity assay to analyze the concentrations of FucGM1 in sera from 112 SCLC patients, 21 patients with non-SCLC, 4 patients with other cancer forms, and 20 healthy controls. Sera were collected at the time of diagnosis before initiation of chemotherapy. The expression of FucGM1 was related to age, sex, blood group of the patient, and to the stage of disease and organ site involvement of metastases. The sera of 50% of the patients with SCLC were positive for FucGM1, and 12 of 21 sera from non-SCLC patients were markedly elevated. In SCLC sera, the concentration of FucGM1 in positive sera ranged from 7 to more than 3000 ng/ml FucGM1. None of 20 controls were positive. FucGM1 correlated to organ site involvement of metastases (p = 0.0016). The ganglioside was detected both at significantly higher concentrations (p = 0.0005) and in significantly more patients (p = 0.0026) with metastases to both the liver and bone marrow, compared to patients with metastases to the liver only.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly of the T-cell antigen receptor. Participation of the CD3 omega chain.

The human TCR is composed of the Ti alpha beta heterodimer in association with the CD3 chains CD3 gamma delta epsilon zeta 2. Another chain, referred to as CD3 omega, has recently been described in T cells. CD3 omega is an intracellular protein transiently associated with the CD3 complex during the assembly of the TCR in the endoplasmic reticulum (ER) and it is not expressed on the cell surface. The function of CD3 omega is unknown but it has been suggested that it plays an important role in the assembly of the TCR. We have studied the possible function of CD3 omega in the human leukemic T-cell line Jurkat and different variants of this cell line. Cells were metabolically labeled, subjected to lysis, immunoprecipitated, and analyzed by SDS-PAGE. The results indicate that: 1) CD3 omega associates primarily with the CD3 delta epsilon complex; 2) CD3 omega is not associated with single Ti alpha or Ti beta chains, but is present in complexes composed of both the CD3 and the Ti chains; 3) CD3 omega is part of the complete, intracellular receptor complex Ti alpha beta/CD3 gamma epsilon delta omega zeta 2; and 4) CD3 omega dissociates from the Ti/CD3 complex in the ER before maturation of the Ti alpha beta heterodimer. On the basis of these results, we propose a model for the assembly and subunit stoichiometry of the TCR complex which includes the participation of the CD3 omega chain.

Induction of CD3 delta epsilon omega by phorbol 12-myristate 13-acetate.

The effect of phorbol 12-myristate 13-acetate (PMA) on the synthesis, assembly and processing of the components of the T cell receptor (TcR) was studied with special focus on the CD3 omega chain. Treatment of the human leukemic T cell line Jurkat with PMA increased the synthesis of the Ti alpha, CD3 gamma and CG3 zeta chains two- to threefold and the synthesis of Ti beta and CD3 delta epsilon omega complexes five- to sevenfold as assessed by metabolic labeling, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by scanning densitometry. The amount of total assembled TcR complexes increased approximately threefold and the maturation of the TcR was not affected as determined by analysis of oligosaccharide side chain processing in the Golgi apparatus. Activation of Jurkat cells with anti-CD3 monoclonal antibody, calcium ionophore, or mitogenic lectins did not affect the synthesis of the TcR components. In other cells studied (the human leukemic T cell line CEM, a panel of variants of the Jurkat T cell line and peripheral blood mononuclear cells) PMA also increased the synthesis of the TcR components. However, for all cell lines studied the amount of TcR complexes expressed on the cell surface was decreased after 16 h of PMA treatment. Based on these results we propose a role of CD3 omega in retention of TcR complexes. From PMA-treated CEM cells more than 50-fold the amount of CD3 delta epsilon omega complexes was immunoprecipitated as compared to the amount obtained from untreated Jurkat cells, and these observations indicate that the CEM cell line may be a qualified candidate for purification of CD3 omega.

Monoclonal antibodies for diagnosis and potential therapy of small cell lung cancer--the ganglioside antigen fucosyl-GM1.

Lung carcinomas represent a highly heterogenous group of tumors, which includes small cell lung cancer (SCLC). The ganglioside fucosyl-GM1 (FucGM1) was originally identified as a highly selective marker for SCLC. A number of monoclonal antibodies against FucGM1 have been produced against the purified ganglioside, and their specificity validated. With monoclonal antibodies, FucGM1 has been detected in tissues as well as in serum samples from SCLC patients. A sensitive, quantitative assay method for FucGM1 was developed based on scintillation proximity immunoassay (SPA): A specific monoclonal antibody against FucGM1 is bound to immunosorbent particles that contain a fluor. When radiolabelled FucGM1 binds to these particles, the fluor is excited, and the photons emitted can be measured directly in a beta-counter. This sensitive assay for FucGM1 has several potential diagnostic applications: differential diagnosis of SCLC, development of serum assays for diagnosis and monitoring of disease progression, and monitoring of patient response to therapy. The expression of FucGM1 in SCLC cells is heterogeneous, and may depend on the differentiated state of tumor cells. However, monoclonal antibodies specific for FucGM1 may have important future applications for radioimmunoimaging as well as in immunotherapy for SCLC.

Serum immunoassay of a small cell lung cancer associated ganglioside: development of a sensitive scintillation proximity assay.

We here report an enzyme linked immunosorbent assay (ELISA) and a scintillation proximity assay (SPA) for detection of the ganglioside FucGM1 in sera from small cell lung cancer (SCLC) patients. The SPA was more sensitive and reproducible than the ELISA. In this assay, monoclonal antibodies specific for FucGM1 were bound to SPA particles and incubated with labelled FucGM1 and 100 microliters test-serum overnight, and counted in a beta-counter. The sensitivity was 0.2 ng. Seven out of twenty sera from SCLC patients were positive, whereas none of twenty sera from healthy individuals were positive for FucGM1. The SPA was more sensitive than the previously reported HPTLC as well as a direct ELISA.

Gastrin releasing peptide GRP(14-27) in human breast cancer cells and in small cell lung cancer.

Immunoreactivity related to the gastrin-releasing peptide (GRP) precursor was detected in four different human breast cancer cell lines. The amounts and the characteristics in extracts from different breast carcinoma cells were compared with cell extracts from small cell lung cancer (SCLC) cells. Two different radioimmunoassays were employed, directed against the amino acid sequence 14-27 of GRP (IR-GRP) or the 42-53 amino acid sequence at the C-terminal end of the GRP precursor (GRP precursor fragment). In extracts from T47D cells cultured under serum free conditions, IR-GRP coeluted with GRP(14-27) or GRP(18-27) in Sephadex G-50 chromatography. No immunoreactivity was detected in the fractions containing high molecular weight components. In a total of 41 human breast carcinoma biopsies from different postmenopausal patients, IR-GRP was detected by immunohistological staining in 39% of the samples. When the GRP(14-27) peptide was added exogenously to breast cancer and SCLC cell lines under serum-free culture conditions, (3H)-thymidine incorporation was stimulated by GRP(14-27) in the SCLC cell lines. Of the breast cancer cell lines only the T47D cell line responded with an increase in (3H)-thymidine incorporation comparable to the increase observed with SCLC cells. Recently, it has been reported that GRP-like receptors are present in some human breast cancer cell lines, including the T47D cell line studied here. The breast cancer cell line T47D therefore expresses the GRP peptide and the receptor for GRP. The identification of GRP-like receptors on T47D cells is in accordance with our present observation of a growth response to GRP(14-27) as evaluated by increased (3H)-thymidine incorporation.

Immunochemical detection of a small cell lung cancer-associated ganglioside (FucGM1) antigen in serum.

Recently, the ganglioside FucGM1 (Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]-Gal beta 1-4Glc beta 1-1 Cer) was identified as a small cell lung cancer (SCLC) marker both in chemical and histochemical studies. In order to further determine whether the FucGM1 ganglioside is shed from the tumor site and consequently is present in the serum of SCLC patients, we produced a series of new monoclonal antibodies raised against FucGM1 and related glycolipids. Shedding of the FucGM1 ganglioside was studied both in vitro and in vivo using SCLC cell lines and nude mice xenografts of SCLC cells as model systems, and finally immunochemical analyses were performed on serum samples from patients with SCLC. High-performance thin-layer chromatography immunostaining demonstrated the presence of FucGM1 in conditioned culture media obtained from FucGM1-positive SCLC cell lines. Furthermore, tumor extracts of SCLC cell line xenografts in nude mice were positive for the FucGM1 marker, and more importantly the marker was also present in serum samples from these mice. Twenty serum samples were obtained from patients with histologically verified SCLC. Eight patients had localized disease, and the remaining patients had disseminated cancer involving metastases to other organ sites. Sera from 4 of these patients were clearly positive, and 2 additional cases were found to be weakly positive. The positive patient sera were all from patients with extensive disease. Sera from 12 patients with non-SCLC and 20 healthy individuals were all found to be negative. These results clearly establish the FucGM1 glycolipid as a potential serum marker of SCLC for which a sensitive immunoassay should be developed and tested using a larger series of serum samples.

Production of gastrin-releasing peptide-(18-27) and a stable fragment of its precursor in small cell lung carcinoma cells.

The production and postsecretory stability of gastrin-releasing peptide (GRP) and the C-terminal part of the GRP precursor were studied in small cell lung cancer cell lines using RIAs developed against these two parts of the precursor. In three otherwise different cell lines (NIC-H345, NIC-H69, and NIC-H510), similar chromatographic patterns of mainly GRP-(18-27) and some GRP-(14-27) along with large fragments of the C-terminal counterpart of the precursor were found to be stored in the cells. In tissue culture medium, gel filtration chromatography indicated that postsecretory limited proteolysis of the GRP precursor fragments occurred. The amount of accumulated immunoreactivity varied among the three cell lines and between the two parts of the precursor. In medium in which only low amounts of GRP immunoreactivity accumulated, the radiolabeled GRP was degraded rapidly. When incubated with plasma, GRP-(14-27) disappeared within a few hours, whereas the C-terminal precursor fragments were stable. It is concluded that the postsecretory stability of peptides excised from the GRP precursor in small cell lung cancer cells varies under tissue culture conditions, but epitopes in the C-terminal part of the precursor are more stable in plasma than the small GRP peptides and, thus, may serve as a better indicator than GRP itself for expression of the GRP precursor in cancer cells.

Gastrin releasing peptide (GRP) is present in a GRP(1-27) form in anterior pituitary cells of the guinea pig.

Immunohistochemical and chromatographic studies were performed on the guinea pig anterior pituitary gland with an antiserum recognizing an epitope within the gastrin releasing peptide (GRP) carboxyterminal amino acid sequence Val-Gly-His-Leu-Met-NH2. Within the anterior pituitary gland GRP-like immunoreactive cells were identified. The GRP-like immunoreactive cells were distributed heterogenously in the gland, predominantly located in ventral aspects of the anterior pituitary. Intracellularly, the immunoreactivity elements were identified as granula-like structures in the cytoplasma. To further characterize the peptide displaying GRP-like immunoreactivity within the pituitary cells, the GRP-like substances were analyzed by radioimmunoassay and gel filtration chromatography. Using this analytical approach it was determined that the guinea pig pituitary extract contained a peptide with characteristics similar to that of authentic porcine GRP(1-27). Only trace amounts of smaller C-terminal fragments were identified. These results indicate, in contrast to findings in other tissues, the GRP(1-27) is not further degraded into smaller peptide fragments.