Alefacept combined with tacrolimus, mycophenolate mofetil and steroids in de novo kidney transplantation: a randomized controlled trial.

Memory T cells play a central role in mediating allograft rejection and are a rational target for immunosuppressive therapy. Alefacept is a recombinant LFA3/IgG1 fusion protein that reduces the number of memory T cells in both psoriatic lesions and the peripheral circulation of psoriasis patients. This study evaluated the efficacy and safety of alefacept compared with placebo when combined with tacrolimus, mycophenolate mofetil and corticosteroids in de novo renal transplant recipients. Between December 2007 and March 2009 patients were randomized in a double-blind fashion to receive alefacept (n = 105) or placebo (n = 107) for 3 months and were then followed for a further 3 months. The primary efficacy endpoint was the incidence of biopsy-confirmed acute T cell mediated rejection (Banff grade ≥ 1) through Month 6. Memory T cell counts were significantly reduced in the alefacept group from Week 3 to study end compared with placebo. However, there was no significant difference between the alefacept and placebo groups for the primary efficacy endpoint (alefacept, 11.0% vs. placebo, 7.0%, p = 0.3). Patient and graft survival as well as renal function was similar between treatment groups. Safety and tolerability were generally similar between the treatment arms. Malignancy was higher in the alefacept treatment arm.

Safety, tolerability, pharmacokinetics, pharmacodynamics and efficacy of the monoclonal antibody ASK8007 blocking osteopontin in patients with rheumatoid arthritis: a randomised, placebo controlled, proof-of-concept study.

OBJECTIVES: Osteopontin is an extracellular matrix protein with diverse immunomodulatory functions. The authors assessed the safety, tolerability, pharmacokinetics, pharmacodynamics and initial efficacy of the humanised monoclonal antibody ASK8007, which blocks osteopontin. METHODS: In this double-blind, multicentre, combined first-in-man, single-dose escalation (phase I, part A) and proof-of-concept, multiple-dose (phase IIA, part B) study, rheumatoid arthritis (RA) patients with active disease were randomly assigned to receive ASK8007 or placebo intravenously. Safety monitoring, pharmacokinetic and pharmacodynamic analyses and clinical assessments were performed throughout the study. The expression of phenotypic cell markers was evaluated in synovial tissue biopsy samples obtained at baseline and 43 days after initiation of treatment (part B) by immunohistochemistry and digital image analysis. Two co-primary efficacy endpoints were the change from baseline in the disease activity score evaluated in 28 joints (DAS28) and the change from baseline in the number of CD68 synovial sublining macrophages, both assessed on day 43 (part B). RESULTS: ASK8007 was overall safe and well tolerated up to the highest studied dose (20 mg/kg). Quantifiable concentrations of ASK8007 were detected in synovial fluid. No differences were observed for changes from baseline in DAS28 and CD68 sublining macrophages between ASK8007 and placebo-treated patients. Within the ASK8007 treatment group, there were also no apparent clinical responses or changes in sublining macrophages. In addition, ASK8007 treatment did not change other assessed biomarkers. CONCLUSIONS: Osteopontin blockade is well tolerated and not related to safety concerns. These results consistently show that osteopontin blockade is unlikely to induce robust clinical improvement in RA patients.

Perioperative blood transfusions, with or without allogeneic leucocytes, relate to survival, not to cancer recurrence.

BACKGROUND: Perioperative blood transfusions are reported to be related to cancer recurrence and reduced survival. Different underlying mechanisms have been proposed, and allogeneic leucocytes in transfused blood have been suggested to contribute to this phenomenon. METHODS: Packed red cells without buffy coat (PC group) were compared with filtered, leucoreduced, red cells (LD group) in a randomized trial of 697 patients with colorectal carcinoma. Five-year survival and cancer recurrence rates were determined, with special emphasis on the location of recurrence. RESULTS: The intention-to-treat analysis showed a survival rate of 63.6 per cent in the PC group and 65.3 per cent in the LD group (P = 0.69), with recurrence rates of 27.8 and 27.9 per cent respectively. The observational analysis showed a significant difference in survival between transfused and non-transfused patients (59.6 versus 72.9 per cent; P < 0.001). The difference in cancer recurrence rate between transfused and non-transfused patients was not statistically significant (29.8 versus 24.3 per cent; P = 0.13). Local recurrences were more frequent in transfused than non-transfused patients (11.9 versus 7.6 per cent; P = 0.09). CONCLUSION: Leucocyte depletion of perioperative transfused blood has no effect on long-term survival and/or cancer recurrence. Perioperative blood transfusions are associated with impaired survival, but not with cancer recurrence. The slight increase in local recurrence rate in transfused patients appears to be related to complicated, in particular rectal, surgery.

Beneficial effects of leukocyte depletion of transfused blood on postoperative complications in patients undergoing cardiac surgery: a randomized clinical trial.

BACKGROUND: Leukocytes in transfused blood are associated with several posttransfusion immunomodulatory effects. Although leukocytes play an important role in reperfusion injury, the contribution of leukocytes in transfused blood products has not been investigated. To estimate the role and the timing of leukocyte filtration of red cells in cardiac surgery, we performed a randomized study. METHODS AND RESULTS: Patients scheduled for cardiac surgery were randomly allocated to receive either packed cells without buffy coat (PC, n = 306), fresh-filtered units (FF, n = 305), or stored-filtered units (SF, n = 303) when transfusion was indicated. We evaluated the periods of hospitalization and stay at the intensive care unit, and the occurrences of postoperative complications up to 60 days after surgery. The average hospital stay was 10.7 days, of which 3.2 days were in the intensive care unit, without significant differences between the groups. In the PC trial arm, 23.0% of the patients had infections versus 16.9% and 17.9% of the patients in the leukocyte-depleted trial arms (P=.13). Within 60 days, 45 patients had died, 24 patients in the PC trial arm (7.8%), versus 11 (3.6%) and 10 (3.3%) patients in the FF and SF trial arms, respectively (P=.015). CONCLUSIONS: In cardiac surgery patients, especially when more than three blood transfusions are required, leukocyte depletion by filtration results in a significant reduction of the postoperative mortality that can only partially be explained by the higher incidence of postoperative infections in the PC group.

Transfusion of red cells is associated with increased incidence of bacterial infection after colorectal surgery: a prospective study.

BACKGROUND: Several studies suggest that perioperative blood transfusion is a major independent risk factor for postoperative bacterial infections. Transfusion-induced immunosuppression is thought to mediate this effect. STUDY DESIGN AND METHODS: In a randomized clinical trial comprising 697 patients with colorectal cancer, the relationship between two types of red cell components (buffy coat-depleted packed red cells and white cell-reduced [filtered] packed red cells) and postoperative bacterial infections was analyzed. RESULTS: Both types of red cells appeared to be associated with a greater incidence of postoperative infection than was no transfusion (39 vs. 24%, p < 0.01). A dose-response relationship could be demonstrated: the corrected relative risk was 1.6 for 1 to 3 units of red cells and 3.6 for more than 3 units. Multivariate analyses identified the transfusion of red cells and tumor location as the only significant independent risk factors for postoperative bacterial infection. CONCLUSION: Because allogeneic white cells, plasma, microaggregates, citrate, and platelets could be ruled out as risk factors for transfusion-associated postoperative infections, it is hypothesized that the transfusion of red cells is a potentially detrimental factor that transiently impairs the clearance of bacteria by phagocytic cells.

Differential expression of HLA-A and B-alleles on uveal melanoma as determined by immuno-histology.

HLA molecules play an important role in the presentation of antigens to the immune system, including tumor-specific antigens. Uveal melanomas vary in the level of expression of monomorphic HLA molecules. However, since the HLA system is polymorphic and since antigen-presentation may be linked to the expression of specific HLA alleles, the authors wondered whether allelic differences in expression existed on uveal melanomas. In order to test this, tissue sections from 23 uveal melanomas were stained in an indirect immunoperoxidase technique with monoclonal antibodies against monomorphic and polymorphic determinants of HLA molecules. All uveal melanomas showed a high level of expression of the monomorphic determinants of HLA-Class I. The polymorphic HLA-Class I molecules A2, A3, Bw4 and Bw6 varied in expression, with a higher expression of HLA-A than of HLA-B. A low level of expression of both ß2-microglobulin and HLA-B locus products was associated with a large tumor diameter. Expression of HLA-Class II molecules was low (0 to 35%). The observation that expression of the HLA-A allelic products was higher than of the HLA-B subtypes may have implications for the search of tumorspecific peptides for immunotherapeutic use: it may be worthwile to select peptides that specifically bind to HLA-A and not to HLA-B.

Radical resection of locally advanced colorectal cancer.

Multivisceral resection has been accepted as treatment for patients with locally advanced colorectal cancer. Nodal status has recently been claimed to be the most important predictor of survival in patients with such disease, with no survival after 2 years for patients with lymph node metastasis. A retrospective analysis was carried out of the prognostic significance of different tumour characteristics, and whether a more limited palliative resection is warranted in patients with positive lymph nodes. Of 1346 patients with colorectal adenocarcinoma operated on between 1987 and 1991, all those with a tumour staged as T4N0M0 or T4N1M0 (94 patients) were selected. From the remainder, 195 patients with stage T3N0M0 and T3N1M0 lesions were randomly selected as a control group. Overall survival was assessed at the beginning of 1993. The most important predictors of survival were lymph node status and involvement of the resection margins of the tumour. Overall survival in patients with T4 tumours who underwent radical resection was not significantly different from that in those with T3 tumours, even in N1 stages. Extended resection did not induce unacceptable morbidity or mortality. Surgery for locally advanced colorectal adenocarcinoma should result in tumour-free margins, and should therefore include multivisceral resection, even in patients with lymph node metastasis.

Antibodies against p53 are associated with poor prognosis of colorectal cancer.

Mutation of the p53 gene is a common event in colorectal cancer. This alteration can result in cellular accumulation of p53 and may also induce p53 antibodies. Accumulation of p53 in tumour cells has been associated with poor prognosis of colorectal cancer. We tested preoperative sera from 255 patients with colorectal cancer by enzyme-linked immunosorbent assay (ELISA). A total of 70.2% had reactivity that was higher than the 'low' control serum. Employing a cut-off level of 10% of the 'high' control sample, 25.5% of the patients were positive for p53 antibodies. The presence of p53 antibodies correlated with the following prognostic factors: histological differentiation grade, shape of the tumour, and tumour invasion into blood vessels. Patients with p53 antibodies were shown to have decreased survival and decreased disease-free survival. Specifically for patients with cancer stage A and B1 the presence of p53 antibodies selected a subgroup with poor prognosis.

A computer program for predicting possible cytotoxic T lymphocyte epitopes based on HLA class I peptide-binding motifs.

Vaccination with peptides recognized by antigen-specific CTLs can prevent lethal virus infections and tumor growth. In order to avoid the synthesis and testing of the numerous overlapping peptide of long AA sequences of proteins of interest, we developed a computer program which utilizes the rules, "motifs" which govern how peptides bind to HLA class I molecules, to derive a predicted binding score for each overlapping peptide. Correlations between the predicted and actual binding results to HLA-A*0201 for 100 peptides selected from six early and two late protein sequences of human papillomavirus type 1a revealed an acceptable level (61%) of concordance. The program is very flexible with regard to the input of protein sequences and motif definitions and is able to handle various motif and peptide lengths.

Randomised controlled trial comparing transfusion of leucocyte-depleted or buffy-coat-depleted blood in surgery for colorectal cancer.

In retrospective studies, perioperative blood transfusions were associated with poor prognosis after surgery for cancer and were a major independent risk factor for postoperative bacterial infection. Leucocyte-depleted, in contrast to buffy-coat-depleted, blood has no immunosuppressive effects in transplantation and so might lack detrimental effects on cancer prognosis and postoperative infections. We studied this hypothesis in a controlled trial by randomly allocating patients to receive either leucocyte-depleted red cells or packed cells without buffy coat when blood was needed. Between 1987 and 1990, 871 eligible patients with colorectal cancer, including 697 patients operated upon with curative intent, were randomised in the 16 participating hospitals. Neither the eligible group nor the curative group showed significant differences between the two trial transfusions in survival, disease-free survival, cancer recurrence rates, or overall infection rates after an average follow-up of 36 months. Patients who had a curative resection and who received blood of any sort had a lower 3-year survival than non-transfused patients (69% vs 81%, p = 0.001) and a higher infection rate (39% vs 24%, p < 0.001). Colorectal cancer recurrence rates, however, were not influenced by blood transfusion (30% vs 26%, p = 0.22). These combined observations confirm the association between blood transfusion and poor patient survival but indicate that the relation is not due to promotion of cancer.

p53, a potential target for tumor-directed T cells.

Cell lineage-specific cellular proteins, oncogenes from viral or cellular origin and tumor suppressor genes encode tumor-specific/associated antigens. Such antigens can elicit an major compatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) response, either naturally in cancer patients or following appropriate immunostimulation (in vitro or in vivo). The reported immune responses in humans to the melanoma-associated MAGE gene products, GP100 and tyrosinase, all self-proteins, support the idea to use wild-type p53 products as targets for T cells. An important step towards this goal is identification of potential p53 CTL epitopes. We identified the wild-type p53 peptides with the highest affinity to the HLA-A*0201 molecule using two assays: the previously described MHC peptide-binding assay and the peptide competition assay. We obtained CTL against four p53 peptides with a high affinity for the HLA-A*0201 molecule. These findings are discussed next to a short review concerning the p53 literature.

The expression of histocompatibility-related leukocyte antigens in the pathway to cervical carcinoma.

The major histocompatibility complex probably plays a crucial role in the efficacy of the cellular immune response against virally infected cervical diseases. Therefore, the allele-specific histocompatibility-related leukocyte antigens (HLA) class I and II expression on normal (n = 10), premalignant (n = 25), and malignant cervical tissue (n = 30) was investigated. No alterations in monomorphic or locus/allele-specific HLA class I or II expression were observed in normal and premalignant epithelial tissue. In cervical carcinomas, however, a reduced expression of HLA class I antigens was present in 70% of the cases, comprising a monomorphic class I loss in 20%, and an allele-specific loss in 50% of HLA-A2-, 66% of A3-, 56% of Bw4-, and 37% of Bw6-positive patients. De novo expression of class II antigens was observed in 80% of the cervical carcinomas, with the sublocus products being expressed in the order HLA-DR > HLA-DQ > HLA-DP. The authors' results show that alteration in HLA expression is a process confined to malignant cells, which may allow tumors to evade immune surveillance. In addition, these findings have to be considered as new strategies of immunotherapy using cytotoxic T lymphocytes are developed.

Association between HLA-expression and infiltration of immune cells in cervical carcinoma.

BACKGROUND: To determine the degree of specificity of the cellular immune response in cervical carcinoma, that is known to be human papillomavirus-related, we investigated the exact relationship between in situ tumor-infiltrating immune cells and the monomorphic/allele-specific HLA expression on the tumor cells. EXPERIMENTAL DESIGN: Attention was focussed on the type, location and number of in situ immunocompetent cells in malignant cervical tissue (N = 30). Immune cell distribution was quantitatively assessed by morphometry for stromal and tumor tissue separately. These results were related to the degree of expression of monomorphic- and allele-specific HLA I and II antigens on the cervical tumor cells. RESULTS: In monomorphic HLA class I downregulated cervical tumors, a significant decrease in tumor-infiltrating CD8+ T cells was observed. However, allele-specific downregulation of respectively HLA-A2, HLA-A3, HLA-Bw4, and HLA-Bw6, did not correlate significantly with a decrease in tumor-infiltrating immune cells. For HLA class II-positive cervical tumors, HLA-DR expression significantly correlated with an increase in the presence of tumor-infiltrating CD3+/CD4+/CD8+ T cells, CD56+ natural killer cells and CD16+ macrophages. No significant correlations were found between alterations in HLA class I or II expression on the tumor cells and stromal infiltrating immune cells. CONCLUSIONS: Our observations provide in situ immunomorphologic evidence that in cervical carcinoma, de novo expression of HLA class II antigens on the tumor cells resulted in an increase of tumor-infiltrating immune cells. In addition, the tumor-infiltrating CD8+ T lymphocytes correlated with monomorphic HLA class I expression on the tumor cells, which stresses the existence of a HLA-restricted immune response of T lymphocytes in cervical carcinoma. These findings might have implications for the biologic behavior of this disease and have to be taken into account in strategies concerning immunotherapy of cervical carcinoma.

In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild-type p53.

The central role of the p53 tumor suppressor gene product in oncogenesis is gradually being clarified. Point mutations in the p53 tumor suppressor gene are common in most human cancers and are often associated with p53 protein overexpression. Overexpressed wild-type or mutant determinants of the p53 protein thus represent an attractive target for immunotherapy of cancer directed against a structure involved in malignant transformation. An important step towards this goal is identification of epitopes of p53 that can be recognized by human cytotoxic T lymphocytes. We identified peptides of (mutant) p53 capable of binding to HLA-A2.1 in an in vitro assay. These HLA-A2.1-binding peptides were utilized for in vitro induction of primary cytotoxic T lymphocyte responses using a human processing-defective cell line (174CEM.T2) as antigen-presenting cell. These cells display "empty" HLA class I surface molecules, that can efficiently be loaded with a single peptide. We obtained CD8+ cytotoxic T lymphocyte clones capable of specifically lysing target cells loaded with wild-type or tumor-specific mutant p53 peptides. This strategy allows the in vitro initiation of human cytotoxic T lymphocyte responses against target molecules of choice.

Characterization of cytotoxic T lymphocyte epitopes of a self-protein, p53, and a non-self-protein, influenza matrix: relationship between major histocompatibility complex peptide binding affinity and immune responsiveness to peptides.

We previously described a motif prediction of major histocompatibility complex allele-specific peptides and an in vitro assay for actual measurement of peptide binding to human leukocyte antigen HLA-A2.1 molecules. Using this method we have identified candidate cytotoxic T lymphocyte (CTL) epitopes derived from a non-self-protein (influenza matrix) and self-protein (p53). We now show that results of binding assays performed over a range of peptide concentrations indicate that distinct differences in HLA-A2.1 peptide binding affinities exist between the influenza matrix and p53 protein. The results for the influenza matrix protein indicate that the peptide that shows the highest binding affinity to HLA-A2.1 is identical to the known immunodominant peptide recognized by influenza virus-specific CTLs. The results for p53 indicate that one of the peptides with a low binding affinity is capable of inducing specific CTL responses, but CTLs recognizing the highest affinity binding peptides were not obtained. These findings are discussed in terms of the distinct implications for induction of cellular immune responses directed against peptides with different binding affinities for HLA-A2.1 of proteins that constitute attractive targets for tumor immunotherapy.

Identification of peptide sequences that potentially trigger HLA-A2.1-restricted cytotoxic T lymphocytes.

We used the human processing defective cell line 174CEM.T2 (T2) to identify potential cytotoxic T lymphocyte (CTL) epitopes of human proteins. Exogenously added peptides can increase the number of properly folded HLA-A2.1 molecules on the cell surface of T2 cells, as shown by immunofluorescence measurements using the mouse monoclonal antibody BB7.2 (anti-HLA-A2.1) and fluorescein isothiocyanate-labeled goat anti-mouse F(ab')2 antibody. The peptides were selected on the basis of a computer score derived from the recently described HLA-A2.1 specific motif. Analysis of the influenza matrix protein showed that 15 out of 35 high-scoring peptides up-regulate the expression of HLA-A2.1 molecules on the T2 cell surface. The combination of the computer scoring program and an immunofluorescence-based peptide binding assay allows rapid detection of potential CTL target peptides.

Prevention of primary cytomegalovirus infection in patients with hematologic malignancies by intensive white cell depletion of blood products.

The effect of white cell depletion of red cells and platelet concentrates on the transmission of cytomegalovirus (CMV) was studied retrospectively in 150 patients treated intensively for acute leukemia or non-Hodgkin's lymphoma. CMV infection was diagnosed on the basis of IgM and IgG antibody responses to CMV late antigen (CMV-LA). Before cytoreductive therapy for their underlying disease, 59 patients were CMV seronegative and 91 were CMV seropositive. None of the 59 CMV-seronegative patients showed persistent seroconversion 2 months after the cytoreductive treatment. The comparison group, consisting of 312 cardiac surgery patients, showed a significantly higher incidence of primary CMV infections: 10 of 86 (11.6%, p = 0.004). Twenty-five percent of the CMV-seronegative patients and controls had transient IgG antibodies to CMV-LA without IgM antibodies, which is indicative of antibodies passively acquired via blood products. These results indicate that white cell-poor blood products carry a very low risk, if any, of CMV transmission. The policy of transfusing white cell-poor blood products provides a useful alternative to the selection of CMV-seronegative donors.