Intraoperative use of ketorolac or diclofenac is associated with improved disease-free survival and overall survival in conservative breast cancer surgery.

BACKGROUND: An association between the use of non-steroidal anti-inflammatory drugs (NSAIDs) and better outcome after mastectomy and lung surgery for cancer has been recently suggested. In a retrospective analysis, we investigated the association between intraoperative NSAIDs use in conservative breast cancer surgery and breast cancer disease-free survival (DFS). Similarly, we also evaluated the association between breast cancer DFS and preoperative neutrophil:lymphocyte ratio (NLR). METHODS: A retrospective analysis of a single-centre cohort was performed in breast cancer patients (n=720) with uni- and multivariate analyses, using a Cox regression model. RESULTS: In conservative breast cancer surgery, the intraoperative use of NSAIDs (ketorolac or diclofenac) was associated with an improved DFS {hazard ratio (HR)=0.57 [95% confidence interval (CI): 0.37-0.89], P=0.01} and an improved overall survival (OS) [HR=0.35 (95% CI: 0.17-0.70), P=0.03]. In these patients, an NLR >3.3 (identified by a receiver-operating characteristic curve) was associated with a shorter DFS [HR=1.99 (95% CI: 1.16-3.41), P=0.01] and OS [HR=2.35 (95% CI: 1.02-5.43), P=0.046]. CONCLUSIONS: Intraoperative NSAIDs and higher preoperative NLR are associated with improved outcome in conservative breast cancer surgery. Prospective, randomized trials to evaluate if these associations are causal are warranted.

Is there a rationale for an anesthesiologist's role against cancer recurrence?

Growth of tumors can accelerate during the peri-operative period. Accordingly, early relapse of cancer occurs in some patients during the first two postoperative years. Temporal and biologic analyses of cancer pathophysiology suggest a link between peri-operative pathophysiological changes and acceleration of tumor growth. Understanding the role of inflammation and its consequences (i.e., immune response, growth factors, dissemination of tumor cells) could lead to define a role of anesthesiologists in reducing cancer recurrence following surgery. We argue for peri-operative pharmacological interventions to reduce cancer relapse, with a focus on non-steroidal anti-inflammatory drugs.

Targets for active immunotherapy against pediatric solid tumors.

The potential role of antibodies and T lymphocytes in the eradication of cancer has been demonstrated in numerous animal models and clinical trials. In the last decennia new strategies have been developed for the use of tumor-specific T cells and antibodies in cancer therapy. Effective anti-tumor immunotherapy requires the identification of suitable target antigens. The expression of tumor-specific antigens has been extensively studied for most types of adult tumors. Pediatric patients should be excellent candidates for immunotherapy since their immune system is more potent and flexible as compared to that of adults. So far, these patients do not benefit enough from the progresses in cancer immunotherapy, and one of the reasons is the paucity of tumor-specific antigens identified on pediatric tumors. In this review we discuss the current status of cancer immunotherapy in children, focusing on the identification of tumor-specific antigens on pediatric solid tumors.

About human tumor antigens to be used in immunotherapy.

The choice of antigens to be used in cancer immunotherapy remains a crucial and difficult issue. This review highlights some properties of the different groups of human tumor antigens recognized by T lymphocytes, focusing on parameters that should influence this choice, such as tumor specificity and level of antigen expression.

Biological and clinical developments in melanoma vaccines.

The identification of antigens recognised on human tumours by autologous T-lymphocytes has opened the way for vaccination strategies involving defined tumour antigens. These vaccinations are therapeutic, i.e. they involve patients with detectable disease. Tumour regressions have been observed in a minority of melanoma patients in Phase I/II trials. Some of these regressions have been complete and long lasting. Improving the efficacy of therapeutic vaccines will critically depend on their capacity to trigger a robust immune response, on the development of appropriate methods to monitor these antitumour immune responses to vaccination and on a better understanding of the mechanisms used by tumours to escape immune attack. Finally, the initiation of large randomised Phase III trials will determine the impact of these vaccines on melanoma treatment.

High-resolution structure of HLA-A*0201 in complex with a tumour-specific antigenic peptide encoded by the MAGE-A4 gene.

The heterotrimeric complex of the human major histocompatibity complex (MHC) molecule HLA-A*0201, beta2-microglobulin and the decameric peptide GVYDGREHTV derived from the melanoma antigen (MAGE-A4 protein has been determined by X-ray crystallography at 1.4 A resolution. MAGE-A4 belongs to a family of genes that are specifically expressed in a variety of tumours. MAGE-A4-derived peptides are presented by MHC molecules at the cell surface to cytotoxic T-lymphocytes. As the HLA-A*0201:MAGE-A4 complex occurs only on tumour cells, it is considered to be an appropriate target for immunotherapy. The structure presented here reveals potential epitopes specific to the complex and indicates which peptide residues could be recognised by T-cell receptors. In addition, as the structure could be refined anisotropically, it was possible to describe the movements of the bound peptide in more detail.

A monoclonal cytolytic T-lymphocyte response observed in a melanoma patient vaccinated with a tumor-specific antigenic peptide encoded by gene MAGE-3.

Vaccination of melanoma patients with tumor-specific antigens recognized by cytolytic T lymphocytes (CTL) produces significant tumor regressions in a minority of patients. These regressions appear to occur in the absence of massive CTL responses. To detect low-level responses, we resorted to antigenic stimulation of blood lymphocyte cultures in limiting dilution conditions, followed by tetramer analysis, cloning of the tetramer-positive cells, and T-cell receptor (TCR) sequence analysis of the CTL clones that showed strict specificity for the tumor antigen. A monoclonal CTL response against a MAGE-3 antigen was observed in a melanoma patient, who showed partial rejection of a large metastasis after treatment with a vaccine containing only the tumor-specific antigenic peptide. Tetramer analysis after in vitro restimulation indicated that about 1/40,000 postimmunization CD8(+) blood lymphocytes were directed against the antigen. The same TCR was present in all of the positive microcultures. TCR evaluation carried out directly on blood lymphocytes by PCR amplification led to a similar frequency estimate after immunization, whereas the TCR was not found among 2.5 x 10(6) CD8(+) lymphocytes collected before immunization. Our results prove unambiguously that vaccines containing only a tumor-specific antigenic peptide can elicit a CTL response. Even though they provide no information about the effector mechanisms responsible for the observed reduction in tumor mass in this patient, they would suggest that low-level CTL responses can initiate tumor rejection.

A point mutation in the alpha-actinin-4 gene generates an antigenic peptide recognized by autologous cytolytic T lymphocytes on a human lung carcinoma.

We have identified an antigen recognized on a human large cell carcinoma by an autologous tumor-specific CTL clone that was derived from mononuclear cells infiltrating the primary tumor. The antigenic peptide is presented by HLA-A2 molecules and is encoded by the alpha-actinin-4 gene, which is expressed ubiquitously. In the tumor cells, a point mutation generates an amino-acid change that is essential for recognition by the CTLS: The mutation was not found in alpha-actinin-4 cDNA sequences from about 50 lung carcinoma cell lines, suggesting that it is unique to this patient. Although he did not receive chemotherapy or radiotherapy, the patient has been without evidence of tumor since the resection of the primary lesion in 1996. Using tetramers of soluble HLA-A2 molecules loaded with the mutated antigenic peptide, anti-alpha-actinin-4 CTLs could be derived from blood samples collected from the patient in 1998 and 2000. It is possible that these CTLs, recognizing a truly tumor-specific antigen, play a role in the clinical evolution of this lung cancer patient.

High frequency of cytolytic T lymphocytes directed against a tumor-specific mutated antigen detectable with HLA tetramers in the blood of a lung carcinoma patient with long survival.

We have identified an antigen recognized by autologous CTL on the lung carcinoma cells of a patient who enjoyed a favorable clinical evolution, being alive 10 years after partial resection of the primary tumor. The antigenic peptide is presented by HLA-A2 molecules and encoded by a mutated sequence in the gene coding for malic enzyme, an essential enzyme that converts malate to pyruvate. In the tumor cell line derived from the patient, only the mutated malic enzyme allele is expressed, because of a loss of heterozygosity in the region of chromosome 6 that contains this locus. Tetramers of soluble HLA-A2 molecules loaded with the antigenic peptide stained approximately 0.4% of the patient's blood CD8 T cells. When these cells were stimulated in clonal conditions, 25% of them proliferated, and the resulting clones were lytic and specific for the mutated malic enzyme peptide. T-cell receptor analysis indicated that almost all of these antimalic CTLs shared the same receptor. Antimalic T cells were consistently found in blood samples collected from the patient between 1990 and 1999, at frequencies ranging from 0.1 to 0.4% of the CD8 cells. Their frequency appeared to double within 2 weeks after intradermal inoculation of lethally irradiated autologous tumor cells. These results indicate that nonmelanoma cancer patients may also have a high frequency of blood CTLs directed against a tumor-specific antigen.

Identification of a tumor-specific shared antigen derived from an Eph receptor and presented to CD4 T cells on HLA class II molecules.

We obtained a lytic CD4 T-cell clone that recognized an antigen presented by HLA-DRB1*1101 on the tumor cells of a melanoma patient who enjoyed an unusually favorable clinical evolution. The antigen appeared to be shared between several melanoma cell lines. To identify the encoding gene, we used a new method, based on the cotransfection into human embryonal kidney cell line 293 of a cDNA library from the tumor together with a cDNA clone encoding the class II transactivator, which induces the expression of HLA class II molecules. The product of the gene coding for the antigenic peptide is EphA3, a member of the Eph family of tyrosine kinase receptors, which mediate the repulsion of neural cells by cells carrying the ligand Ephrins on their surface. EphA3 is expressed at a high level in the retina and fetal brain, at a lower level in several normal tissues, and not at all in hematopoietic cells, the only cells that constitutively express HLA class II molecules. It is overexpressed in several types of tumors, including melanoma, lung carcinoma, and sarcoma. On the basis of this pattern of expression, EphA3 may be a source of tumor-specific antigens recognized on tumor cells that express HLA class II molecules. Anti-EphA3 T cells may have participated in a tumor rejection response in the patient, because the cells of metastases collected several years later than the metastasis used to characterize the antigen had lost expression of HLA-DR or EphA3, therefore escaping recognition by these lymphocytes.

Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A1-MAGE-A1 from a nonimmunized phage-Fab library.

Antitumor antibodies with the same specificity as cytotoxic T lymphocytes that recognize antigenic peptides encoded by tumor-associated genes and presented by MHC class I molecules would be valuable tools to analyze the antigenicity or target tumor cells in vivo. To obtain a human antibody directed against a peptide encoded by gene melanoma-associated antigen (MAGE)-A1 and presented by HLA-A1 molecules, we selected a large phage Fab antibody repertoire on a recombinant version of the complex HLA-A1-MAGE-A1 produced by in vitro refolding. One of the selected phage antibodies shows binding to HLA-A1 complexed with the MAGE-A1 peptide, but does not show binding to HLA-A1 complexed with a peptide encoded by gene MAGE-A3 and differing from the MAGE-A1 peptide by only three residues. Phages carrying this recombinant antibody bind to HLA-A1(+) cells only after in vitro loading with MAGE-A1 peptide. These results indicate that nonimmunized phage Fab libraries are a source of antibodies with a T cell antigen receptor-like specificity. The human anti-HLA-A1-MAGE-A1 antibody described here may prove very useful for monitoring the cell surface expression of these complexes, and eventually, as a targeting reagent for the specific immunotherapy of HLA-A1 patients bearing a MAGE-A1-positive tumor.

Expression of gene MAGE-A4 in Reed-Sternberg cells.

Genes of the MAGE-A family are expressed in several types of solid tumors but are silent in normal tissues with the exception of male germline cells, which do not carry HLA molecules.Therefore, peptides encoded by MAGE-A genes are strictly tumor-specific antigens that can be recognized by CTL and constitute promising targets for immunotherapy. The expression of 6 genes of the MAGE-A family was tested with reverse transcriptase-polymerase chain reaction in lymphoma samples. Among 38 samples of non-Hodgkin lymphoma, 1 anaplastic large cell lymphoma expressed genes MAGE-A1, -A2, -A3, -A4, and -A12, and 1 lymphoepithelioid T-cell lymphoma expressed gene MAGE-A4. Five of 18 samples (28%) from patients with Hodgkin disease expressed gene MAGE-A4. In tissue sections, staining by a monoclonal antibody that recognizes the MAGE-A4 protein was observed in 11 of 53 samples (21%) from patients with Hodgkin disease. In the positive samples, the Reed-Sternberg cells were strongly stained whereas the surrounding cells were not. These results indicate that Hodgkin disease may be a target for specific immunotherapy involving MAGE-A4 antigens.

High frequency of autologous anti-melanoma CTL directed against an antigen generated by a point mutation in a new helicase gene.

We have identified an Ag recognized by autologous CTL on the melanoma cells of a patient who enjoyed an unusually favorable clinical evolution. The antigenic peptide, which is presented by HLA-A28 molecules, is encoded by a mutated sequence in a new gene. This gene, which was named MUM-3, is expressed ubiquitously and shows homology with the RNA helicase gene family. Limiting dilution analysis indicated that at least 0.15% of the blood CD8 T cells were tumor-specific CTL precursors. The MUM-3 Ag was recognized by 90% of these CTL, indicating that it is the dominant target Ag of the tumor-specific CTL response. The high frequency of anti-MUM-3 CTL was confirmed with tetramers of soluble HLA-A28 molecules loaded with the antigenic peptide. MUM-3 tetramers stained 1.2% of blood CD8 cells, a frequency that has never been reported for T cells directed against a strictly tumor-specific Ag. To confirm these results, the CD8 T cells that were clearly labeled with tetramers were restimulated in clonal conditions. About 90% of these cells proliferated, and all the resulting clones proved lytic and MUM-3 specific. By improving the conditions used for the in vitro restimulation of CTL precursors by the tumor cells, the same frequency could be obtained in limiting dilution analysis. These results show that some cancer patients have a high frequency of circulating CTL that are directed against a strictly tumor-specific Ag. These CTL are responsive to restimulation in vitro and are easily detected with tetramers. Such responses may therefore be an achievable goal for therapeutic vaccination with tumor-specific Ags.

Analysis of the Fv1 alleles involved in the susceptibility of mice to lactate dehydrogenase-elevating virus-induced polioencephalomyelitis.

Development of polioencephalomyelitis in mice infected with lactate dehydrogenase-elevating virus (LDV) requires expression of N-tropic ecotropic MuLV retroviruses. 129/Sv mice are resistant to N-tropic MuLV expression and therefore do not develop LDV-induced polioencephalomyelitis. The Fv1 gene determines the susceptibility to retrovirus replication. We sequenced the open reading frame of the Fv1nr allele of 129/Sv mice. It differs by only one nucleotide, modifying one amino acid in the encoded protein, from the Fv1n allele of susceptible AKR and C58 animals. We excluded that the resistance of 129/Sv mice to LDV-induced polioencephalomyelitis resulted from the absence of endogenous N-tropic retrovirus, by infecting (129/Sv x C58/J) F1 animals. Therefore it is possible that the amino acid that defines the Fv1nr allele is responsible for resistance of 129/Sv mice to N-tropic MuLV expression and to LDV-induced polioencephalomyelitis.

Two antigens recognized by autologous cytolytic T lymphocytes on a melanoma result from a single point mutation in an essential housekeeping gene.

We have pursued our analysis of antigens recognized by autologous cytolytic T lymphocytes (CTLs) on the melanoma cells of patient LB33. This patient enjoys an unusually favorable evolution, which is associated with a strong and sustained antitumor CTL response. We reported previously the analysis of two melanoma cell lines, MEL.A and MEL.B, which were derived from metastases removed from the patient at 5 years' distance. Autologous CTL clones derived from blood lymphocytes recognized several antigens presented by different HLA class I molecules on MEL.A. The MEL.B cells resisted lysis by these CTLs because they have lost expression of most HLA molecules, suggesting that they were selected in vivo by the anti-MEL.A CTL response. One of the MEL.A antigens was shown to result from a point mutation in the tumor. Here we report the cloning of a gene that encodes two other MEL.A antigens. This new gene, MUM-2, is expressed ubiquitously. In the melanoma cells of patient LB33, it contains a point mutation that changes one amino acid in the translated protein. Two different antigenic peptides, one presented to CTL by HLA-B44 molecules and another by HLA-C6 molecules, overlap and contain the mutated residue. Gene MUM-2 is homologous to an essential yeast gene, bet5, that was recently shown to be implicated in the vesicular transport of proteins from the endoplasmic reticulum to the Golgi. In a mutant yeast with a disrupted bet5 gene, both the wild-type and the mutated MUM-2 genes could complement for bet5 function. These results indicate that the antigenic mutation does not destroy the function of the protein, a function that is conserved in eukaryotic cells. The identification of these antigens suggests that point mutations could be the major cause of the strong immunogenicity of MEL.A cells.

Correction of HLA-Cw*0501 and identification of HLA-Cw*0711.

We describe a new HLA-C allele, Cw*0711, that differs from Cw*0704 by one residue in the intracytoplasmic region, and correct two errors in the published sequence of Cw*0501.

Antitumor immunity at work in a melanoma patient.

This review covers the results obtained so far with a chronological analysis of the antitumor cytolytic T lymphocyte (CTL) cell response of a melanoma patient who enjoys an unusually favorable evolution. Two melanoma cell lines, MEL.A and MEL.B, were derived from metastases removed from the patient in 1988 and 1993, respectively. The patient developed a very strong CTL response against the MEL.A cells. Several antigens on these cells, presented by various HLA class I molecules, result from point mutations present in the genome of the tumor. The MEL.B cells, on the other hand, resist lysis by these CTLs because they have lost expression of most HLA class I molecules, suggesting that they were selected in vivo by the anti-MEL.A CTLs. New CTLs recognize MEL.B cells specifically, however. Analysis of their specificity indicates that they carry inhibitory receptors similar to those present on natural killer (NK) cells. These results illustrate the relationship between a tumor and the immune system in vivo over a period of several years. They are discussed in the context of the recent identification of many human tumor antigens recognized by CTLs, and the perspectives of specific immunotherapy opened up by these discoveries.

Differences in the recognition by CTL of peptides presented by the HLA-B*4402 and the HLA-B*4403 molecules which differ by a single amino acid.

The HLA-B*4402 and B*4403 molecules differ only at residue 156, which borders the peptide binding site. Strong in vivo allogeneic reactions mediated by cytolytic T lymphocytes (CTLs) were reported in patients who received a bone marrow graft mismatched for these B44 subtypes, indicating that HLA-B*4402 and B*4403 molecules present distinct antigens. This could be due either to the presentation of different sets of antigenic peptides or to the recognition by CTLs of conformational epitopes formed by the MHC molecules alone or in association with antigenic peptides. To address this question, we compared the two B44 subtypes in their presentation to tumor-specific CTLs of three peptides, encoded by genes MAGE-3, MUM-1 and Tyrosinase. The peptides bound with similar affinities to B*4402 or B*4403 molecules, as assessed by lytic competition assays. One HLA-B*4402-restricted and one HLA-B*4403-restricted CTL clone were derived against each peptide. When tested for lysis of B*4402 and B*4403 cells incubated with the antigenic peptides, most CTLs showed a marked preference for one of the two B44 subtypes. Using variant peptides incorporating single alanine substitutions, we compared a given CTLs' recognition of its antigenic peptide presented by both B44 subtypes. Some substitutions, which had no effect on the binding of the peptide, affected its recognition by the same CTL differently on B*4402 and B*4403 molecules. These results imply that the conformations adopted by the same peptide on the two HLA-B44 subtypes are different. We conclude that the B44 subtype specificity of T cells results mostly from distinct conformations adopted by the same peptides in the two B44 molecules. This does not exclude the possibility that in some cases the B44 subtype specificity results from the selective binding of a peptide to one subtype. We found several peptides, different from the three mentioned above, that contain the canonical HLA-B44 binding motif and bind to B*4403 but not to B*4402 molecules.

Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1.

Thirty-nine tumor-bearing patients with metastatic melanoma were treated with 3 subcutaneous injections of the MAGE-3.A1 peptide at monthly intervals. No significant toxicity was observed. Of the 25 patients who received the complete treatment, 7 displayed significant tumor regressions. All but one of these regressions involved cutaneous metastases. Three regressions were complete and 2 of these led to a disease-free state, which persisted for more than 2 years after the beginning of treatment. No evidence for a cytolytic T lymphocyte (CTL) response was found in the blood of the 4 patients who were analyzed, including 2 who displayed complete tumor regression. Our results suggest that injection of the MAGE-3.A1 peptide induced tumor regression in a significant number of the patients, even though no massive CTL response was produced.