Eva Rossmann

Durable Carcinoembryonic Antigen (CEA)-Specific Humoral and Cellular Immune Responses in Colorectal Carcinoma Patients Vaccinated with Recombinant CEA and Granulocyte/Macrophage Colony-Stimulating Factor

Purpose: Previous studies have indicated that carcinoembryonic antigen (CEA) might be a suitable immunotherapeutic target in colorectal carcinoma (CRC). The aim of the present study was to analyze the immunological and clinical effects of vaccination with CEA together with the adjuvant granulocyte/macrophage colony-stimulating factor (GM-CSF). Experimental Design: Twenty-four resected CRC patients without macroscopic disease were immunized seven times with recombinant CEA at four different dose levels over a 12-month period. Half of the patients received GM-CSF (80 μg/day for 4 consecutive days) at each immunization. Patients were monitored immunologically for 36 months and clinically for 76 months. T-cell response was evaluated by a [3H]thymidine incorporation assay, and IgG response was determined by ELISA. Results: Minor local side effects were common. All 12 patients (100%) in the GM-CSF group developed a CEA-specific T-cell as well as an IgG response. The corresponding figures in the CEA alone group were 9 of 12 (75%) and 8 of 12 (66%), respectively. GM-CSF significantly augmented the amplitude of the T-cell response and the IgG titers. No dose–response relationship was noted. The immune responses at 12 months persisted 24 months after the last vaccination. Anti-CEA IgG titers were associated with increased survival (P < 0.05), whereas standard prognostic factors had no relationship, with the exception of serum CEA value. Conclusions: Vaccination with recombinant CEA and GM-CSF appears to be a nontoxic regimen inducing potent and durable antigen-specific IgG and T-cell response. The results of this study justify more extensive trials with recombinant CEA protein for immunotherapy of CRC.

Signalling molecules and cytokine production in T cells of multiple myeloma‐increased abnormalities with advancing stage

T‐cell immune dysfunction in patients with malignant tumours has been attributed to the altered expression of components of the T‐cell receptor (TCR)/CD3 complex and their associated intracellular protein tyrosine kinases. In this study, four‐colour flow cytometry was applied to study the surface bound molecules TCRαβ, CD28, CD152 and CD154 involved in T‐cell signalling and the signal transduction molecules CD3ζ, p56lck, p59fyn, ZAP‐70 and phosphatidyl‐inositol‐3 kinase (PI3‐k) as well as the intracellular cytokines interferon‐γ (IFN‐γ), interleukin (IL)‐4 and IL‐2 as a functional read‐out of non‐stimulated and superantigen (staphylococcus enterotoxin B)‐stimulated blood T cells of multiple myeloma (MM) patients at different stages of the disease. Multiple abnormalities were demonstrated in the CD4 and CD8 populations, both under non‐stimulated and superantigen‐stimulated conditions. There was a marked reduction, particular in advanced stage MM, in the proportion of CD4 and CD8 cells expressing CD28, CD152, CD3ζ, p56lck, ZAP‐70 and PI3‐k. The level of intracellular T‐cell cytokines (IFN‐γ, IL‐2 and IL‐4) was normal or increased in non‐stimulated cells but activation‐induced cytokine production was impaired. These results illustrated profound and multiple T‐cell signalling defects, from the surface and down‐stream, consistent with involvement of a master T‐cell function, especially in advanced stage MM. These data should be taken into consideration when developing immune‐based therapeutic approaches and when applying new emerging technologies that aim to restore T‐cell functions.

Intracellular T cell cytokines in patients with B cell chronic lymphocytic leukaemia (B‐CLL)

Abstract: Analysis of cytokine production is a tool to functionally characterise T cells. In this study, spontaneous and polyclonal activation induced cytokine production in T cells were assessed by flow cytometry in patients with B‐CLL. Patients with progressive disease had a significantly increased number of T cells spontaneously producing IL‐2, IL‐4 and GM‐CSF as compared to healthy donors and patients with non‐progressive CLL, which was not the case for TNF‐α and IFN‐γ producing T cells. However, no difference in the frequency of T cells producing these cytokines was seen comparing patients with non‐progressive disease to control donors. Polyclonal activation of B‐CLL T cells in vitro induced an increased proportion of T cells producing these five cytokines in patients as well as in control donors, indicating that T cells in CLL patients might have a relatively well preserved functional capacity. However, the increase in GM‐CSF, TNF‐α and IL‐4 producing T cells was more marked in CLL patients than in controls. Furthermore, following activation, a higher frequency of cytokine‐producing T cells was noted in patients with progressive disease as compared to those with non‐progressive disease. The augmented number of cytokine‐producing T cells in CLL may indicate an up‐regulated capability of T cells to secrete cytokines, especially in patients with progressive CLL. The increased production of the T cell derived cytokines GM‐CSF, TNF‐α, IL‐4 and IL‐2 is interesting, as these cytokines have previously been shown to support growth of B‐CLL leukaemic cells in vitro and as T cells might specifically recognise the autologous leukaemic B cells in vivo. The findings may suggest a role for T cells in the pathogenesis of B‐CLL.

Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemia

The fluorescence intensities of CD3, CD4 on T cells and CD20, CD22 molecules on B cells were quantitatively measured on lymphocytes from chronic lymphocytic leukemia (CLL) patients and healthy donors.

Significance of phosphotyrosine proteins, Bcl-2 and p53 for apoptosis in resting b-chronic lymphocytic leukemia (CLL) cells

Signal transduction and apoptosis in B‐cell chronic lymphocytic leukemia (CLL) cells with a post‐germinal center (GC) phenotype were studied. Specific activation of the cells was induced by a combination of soluble anti‐CD40 monoclonal antibody and interleukin‐4 (CD40/IL‐4) and nonspecific activation with a combination of phytohemagglutinin, phorbol‐12‐myristate‐13‐acetate and ionomycin (chemical mixture). Less than 5% of these leukemia cells entered the cell cycle after activation, as indicated by the number of cells in G0/G1 phase. The protein tyrosine phosphorylation pattern and expression of the Bcl‐2 protein were specific in ex vivo CLL cells of each individual patient. Expression of the p53 protein was not detectable in these leukemia cells. Cross‐linking of the CD40/IL‐4 receptors on CLL cells significantly upregulated phosphotyrosine proteins and the p53 protein. In the presence of chemical mixture, downregulated phosphotyrosine proteins were detected. Alterations in Bcl‐2 expression were independent of cross‐linking with CD40/IL‐4 or chemical mixture. A high frequency of apoptotic cells was detected in cells that had downregulated phosphotyrosine proteins and Bcl‐2 protein. There was no correlation between induction of apoptosis and expression of p53 protein. Our results suggest that apoptosis in resting leukemia cells could occur prior to the cell cycle progression. Alterations in phosphotyrosine proteins and Bcl‐2 but not p53 might play an important role in the regulation of apoptosis in resting G0/G1 memory post‐GC B‐CLL cells.

Mucin 1-specific active cancer immunotherapy with tecemotide (L-BLP25) in patients with multiple myeloma: an exploratory study.

Patients (n = 34) with previously untreated, slowly progressive asymptomatic stage I/II multiple myeloma or with stage II/III multiple myeloma in stable response/plateau phase following conventional anti-tumor therapy were immunized repeatedly with the antigen-specific cancer immunotherapeutic agent tecemotide (L-BLP25). Additionally, patients were randomly allocated to either single or multiple low doses of cyclophosphamide to inhibit regulatory T cells (Treg). Immunization with tecemotide resulted in the induction/augmentation of a mucin 1-specific immune response in 47% of patients. The immune responses appeared to involve a Th1-like cellular immune response involving CD4 and CD8 T cells. The rate of immune responses was similar with single versus multiple dosing of cyclophosphamide and in patients with vs. without pre-existing mucin 1 immunity. On-treatment reductions in the slope of M-protein concentration over time (but not fulfilling clinical criteria for responses with conventional anti-tumor agents) were observed in 45% of evaluable patients, predominantly in those without versus with pre-existing mucin 1 immunity and in patients with early stage disease. No differences were seen in patients receiving single or multiple cyclophosphamide dosing. Treatment with tecemotide was generally well tolerated. Repeated vs. single dosing of cyclophosphamide had no impact on Treg numbers and was stopped after a case of fatal encephalitis that was assessed as possibly study-related. Tecemotide immunotherapy induces mucin 1-specific cellular immune responses in a substantial proportion of patients, with preliminary evidence of changes in the M-protein concentration time curve in a subset of patients.

Phase I study of lenalidomide and alemtuzumab in refractory chronic lymphocytic leukaemia: maintaining immune functions during therapy-induced immunosuppression

R., Stevenson, F. & Packham, G. (2003) Differential signaling via surface IgM is associated with VH gene mutational status and CD38 expression in chronic lymphocytic leukemia. Blood, 101, 1087–1093. Traverse-Glehen, A., Davi, F., Ben Simon, E., CalletBauchu, E., Felman, P., Baseggio, L., Gazzo, S., Thieblemont, C., Charlot, C., Coiffier, B., Berger, F. & Salles, G. (2005) Analysis of VH genes in marginal zone lymphoma reveals marked heterogeneity between splenic and nodal tumors and suggests the existence of clonal selection. Haematologica, 90, 470–478. Wierda, W.G., Castro, J.E., Aguillon, R., Sampath, D., Jalayer, A., McMannis, J., Prussak, C.E., Keating, M. & Kipps, T.J. (2010) A phase I study of immune gene therapy for patients with CLL using a membrane-stable, humanized CD154. Leukemia, 24, 1893–1900.

A Phase I Dose-Escalation Study of Lenalidomide in Combination with Gemcitabine in Patients with Advanced Pancreatic Cancer

Purpose Lenalidomide have both immunomodulatory and anti-angiogenic properties which could confer anti-cancer effects. The aim of this study was to assess the feasibility of combining lenalidomide with the standard treatment gemcitabine in pancreatic cancer patients with advanced disease. Patients and Methods Eligible patients had locally advanced or metastatic adenocarcinoma of the pancreas. Patients received lenalidomide days 1–21 orally and gemcitabine 1000 mg/m2 intravenously (days 1, 8 and 15), each 28 day cycle. Three cohorts of lenalidomide were examined (Cohort I = 15 mg, Cohort II = 20 mg and Cohort III = 25 mg daily). The maximum tolerated dose (MTD) of lenalidomide given in combination with gemcitabine was defined as the highest dose level at which no more than one out of four (25%) subjects experiences a dose-limiting toxicity (DLT). Patients should also be able to receive daily low molecular weight heparin (LMWH) (e.g. dalteparin 5000 IU s.c. daily) as a prophylactic anticoagulant for venous thromboembolic events (VTEs). Twelve patients (n = 4, n = 3 and n = 5 in cohort I, II and III, respectively) were enrolled in this study. Results Median duration of treatment was 11 weeks (range 1–66), and median number of treatment cycles were three (range 1–14). The only DLT was a cardiac failure grade 3 in cohort III. Frequent treatment-related adverse events (AEs) (all grades) included neutropenia, leucopenia and fatigue (83% each, but there was no febrile neutropenia); thrombocytopenia (75%); dermatological toxicity (75%); diarrhea and nausea (42% each); and neuropathy (42%). Discussion This phase I study demonstrates the feasibility of the combination of lenalidomide and gemcitabine as first-line treatment in patients with advanced pancreatic cancer. The tolerability profile demonstrated in the dose escalation schedule of lenalidomide suggests the dosing of lenalidomide to be 25 mg daily on days 1–21 with standard dosing of gemcitabine and merits further evaluation in a phase II trial. Trial Registration ClinicalTrials.gov NCT01547260

HAX-1 overexpression in multiple myeloma is associated with poor survival

HS-1 associated protein X-1 (HAX-1/HAX1) was identified 20 years ago as a protein that interacts with HS-1, a substrate of Src family tyrosine kinases. HAX-1 has been shown to be involved in multiple cellular processes, including apoptosis, calcium homeostasis and cell migration (Fadeel & Grzybowska, 2009). Homozygous mutations with loss of HAX-1 expression are seen in patients with congenital neutropenia characterized by excessive apoptosis of myeloid progenitor cells (Klein et al, 2007). HAX-1 was also found to regulate apoptosis in Epstein– Barr virus-transformed human B cell lines (Jitkaew et al, 2009) and cleavage of HAX-1 has been implicated in the propagation of mitochondria-dependent apoptosis (Han et al, 2010). We previously reported that HAX-1 is highly expressed at the mRNA and protein level in B lymphoma, including diffuse large B cell lymphoma and Burkitt lymphoma (Kwiecinska et al, 2011). Moreover, Baumann et al (2014) identified somatic stabilizing mutations of HAX1 in patients with mantle cell lymphoma (MCL) and suggested that HAX1 is a protooncogene in MCL. Multiple myeloma (MM) is a late B cell/ plasma cell malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow, increased monoclonal immunoglobulin in serum and lytic bone destruction. The introduction of novel drugs, including proteasome inhibitors, has increased response rates and survival in recent years, but MM still remains incurable and most patients ultimately relapse due to the presence of residual tumour cells. There are no studies to date of HAX-1 expression or function in MM. Using the public in silico transcriptomics (IST) database, we observed elevated expression of HAX1 in MM as well as in plasma cell leukaemia (Fig 1A). In fact, HAX1 expression was higher in MM than in all other malignancies in the IST database (Kilpinen et al, 2008; http://ist.medisapiens.com), including B lymphomas. Next, we examined the expression of HAX-1 protein in a series of paraffin-embedded diagnostic samples from 41 previously untreated MM patients (Table SI) retrieved from the archives of the Department of Pathology and Cytology at the Karolinska University Hospital, Stockholm. The diagnosis was established according to the International Myeloma Working Group criteria (Rajkumar et al, 2014). HAX-1 protein was found to be highly expressed in 54% of the cases (22/41). Fifteen samples displayed weak or heterogeneous expression in a high fraction of myeloma cells while the remaining samples were negative for HAX-1 or showed weak expression in only a few cells. HAX-1 expression displayed a granular pattern, consistent with the known mitochondrial localization of HAX-1, while a diffuse cytoplasmic expression was noted in a few cases. Representative immunohistochemical staining patterns are shown in Fig 1B–D. Semiquantitative evaluation of HAX-1 expression was performed as previously described (Kwiecinska et al, 2011) and we then investigated whether HAX-1 expression was associated with clinical outcome (Table SI). Survival curves were drawn using the Kaplan-Meier method (Fig 1E). Our analysis indicated a significant difference in survival between MM patients with high and weak expression of HAX-1 (P = 0 002). To elucidate the potential role of HAX-1 in MM, we performed in vitro experiments using the human MM cell lines, U266 and RPMI8226, obtained from the American Tissue Culture Collection (Manassas, VA, USA). HAX-1 protein expression was observed in both MM cell lines; primary human CD19 B cells were included for comparison (Fig 2A). We then transfected U266 and RPMI8226 cells with siRNA against