Immunoscore and Immunoprofiling in cancer: an update from the melanoma and immunotherapy bridge 2015.

The fifth "Melanoma Bridge Meeting" took place in Naples, December 1-5th, 2015. The main topics discussed at this meeting were: Molecular and Immuno advances, Immunotherapies and Combination Therapies, Tumor Microenvironment and Biomarkers and Immunoscore. The natural history of cancer involves interactions between the tumor and the immune system of the host. The immune infiltration at the tumor site may be indicative of host response. Significant correlations were shown between the levels of immune cell infiltration in tumors and patient's clinical outcome. Moreover, incredible progress comes from the discovery of mutation-encoded tumor neoantigens. In fact, as tumors grow, they acquire mutations that are able to influence the response of patients to immune checkpoint inhibitors. It has been demonstrated that sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. The road ahead is still very long, but the knowledge of the mechanisms of immune escape, the study of tumor neo-antigens as well as of tumor microenvironment and the development of new immunotherapy strategies, will make cancer a more and more treatable disease.

IRF-1 responsiveness to IFN-γ predicts different cancer immune phenotypes.

BACKGROUND: Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes. METHODS: IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both. Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation. RESULTS: We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment. Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts. Functional interpretation analysis showed mTOR and Wnt/ß-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis. CONCLUSION: Our findings support the central role of IRF-1 in influencing different tumour phenotypes.

CXCR3/CCR5 pathways in metastatic melanoma patients treated with adoptive therapy and interleukin-2.

BACKGROUND: Adoptive therapy with tumour-infiltrating lymphocytes (TILs) induces durable complete responses (CR) in ∼20% of patients with metastatic melanoma. The recruitment of T cells through CXCR3/CCR5 chemokine ligands is critical for immune-mediated rejection. We postulated that polymorphisms and/or expression of CXCR3/CCR5 in TILs and the expression of their ligands in tumour influence the migration of TILs to tumours and tumour regression. METHODS: Tumour-infiltrating lymphocytes from 142 metastatic melanoma patients enrolled in adoptive therapy trials were genotyped for CXCR3 rs2280964 and CCR5-Δ32 deletion, which encodes a protein not expressed on the cell surface. Expression of CXCR3/CCR5 in TILs and CXCR3/CCR5 and ligand genes in 113 available parental tumours was also assessed. Tumour-infiltrating lymphocyte data were validated by flow cytometry (N=50). RESULTS: The full gene expression/polymorphism model, which includes CXCR3 and CCR5 expression data, CCR5-Δ32 polymorphism data and their interaction, was significantly associated with both CR and overall response (OR; P=0.0009, and P=0.007, respectively). More in detail, the predicted underexpression of both CXCR3 and CCR5 according to gene expression and polymorphism data (protein prediction model, PPM) was associated with response to therapy (odds ratio=6.16 and 2.32, for CR and OR, respectively). Flow cytometric analysis confirmed the PPM. Coordinate upregulation of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumour biopsies was associated with OR. CONCLUSION: Coordinate overexpression of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumours was associated with responsiveness to treatment. Conversely, CCR5-Δ32 polymorphism and CXCR3/CCR5 underexpression influence downregulation of the corresponding receptors in TILs and were associated with likelihood and degree of response.

Epstein-Barr virus microRNAs and lung cancer.

BACKGROUND: We conducted the first analysis of viral microRNAs (miRNAs) in lung cancer, with a focus on Epstein-Barr virus (EBV). METHODS: We evaluated viral miRs with a two-channel oligo-array targeting mature, anti-sense miRNAs in 290 cases. In 48 cases, we compared microarray and real-time quantitative PCR (qPCR) expression for three EBV miRNAs. We tested for EBV DNA, RNA, and protein in tumour tissue from six cases with and six cases without strong qPCR-based evidence of EBV miRNAs. RESULTS: The EBV miRNAs strongly differentiated between adenocarcinoma and squamous cell carcinoma using the microarray (P<0.01 for 9 out of 16 EBV miRNAs). However, microarray and qPCR measurements of BART1, BART2, and BHRF1-3 expression were not significantly correlated (P=0.53, 0.94, and 0.47, respectively). Although qPCR provided substantial evidence of EBV miRNAs in 7 out of 48 cases, only 1 of these 7 cases had detectable EBV DNA in tumour tissue. None had detectable EBV RNA or protein by histochemical stains. CONCLUSION: In a comprehensive evaluation of EBV miRNA, DNA, RNA, and protein in lung cancer, we found little evidence of EBV in lung tumour tissue. Discrepancies between microarray- and qPCR-based strategies highlight the difficulty of validating molecular markers of disease. Our results do not support a role of EBV in lung cancer.

Cancer therapy using a self-replicating RNA vaccine.

'Naked' nucleic acid vaccines are potentially useful candidates for the treatment of patients with cancer, but their clinical efficacy has yet to be demonstrated. We sought to enhance the immunogenicity of a nucleic acid vaccine by making it 'self-replicating'. We accomplished this by using a gene encoding an RNA replicase polyprotein derived from the Semliki forest virus, in combination with a model antigen. A single intramuscular injection of a self-replicating RNA immunogen elicited antigen-specific antibody and CD8+ T-cell responses at doses as low as 0.1 microg. Pre-immunization with a self-replicating RNA vector protected mice from tumor challenge, and therapeutic immunization prolonged the survival of mice with established tumors. The self-replicating RNA vectors did not mediate the production of substantially more model antigen than a conventional DNA vaccine did in vitro. However, the enhanced efficacy in vivo correlated with a caspase-dependent apoptotic death in transfected cells. This death facilitated the uptake of apoptotic cells by dendritic cells, providing a potential mechanism for enhanced immunogenicity. Naked, non-infectious, self-replicating RNA may be an excellent candidate for the development of new cancer vaccines.

Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma.

The cloning of the genes encoding cancer antigens has opened new possibilities for the treatment of patients with cancer. In this study, immunodominant peptides from the gp100 melanoma-associated antigen were identified, and a synthetic peptide, designed to increase binding to HLA-A2 molecules, was used as a cancer vaccine to treat patients with metastatic melanoma. On the basis of immunologic assays, 91% of patients could be successfully immunized with this synthetic peptide, and 13 of 31 patients (42%) receiving the peptide vaccine plus IL-2 had objective cancer responses, and four additional patients had mixed or minor responses. Synthetic peptide vaccines based on the genes encoding cancer antigens hold promise for the development of novel cancer immunotherapies.

Selective histocompatibility leukocyte antigen (HLA)-A2 loss caused by aberrant pre-mRNA splicing in 624MEL28 melanoma cells.

Histocompatibility leukocyte antigen (HLA)-A2 is used as a restricting element to present several melanoma-associated antigen (MAA)-derived peptides to cytotoxic T lymphocytes (CTLs). HLA-A2 antigen is selectively lost in primary melanoma lesions and more frequently in metastases. Only scanty information is available about the molecular mechanisms underlying this abnormality, in spite of its potentially negative impact on the clinical course of the disease and on the outcome of T cell-based immunotherapy. Therefore, in this study we have shown that the selective HLA-A2 antigen loss in melanoma cells 624MEL28 is caused by a splicing defect of HLA-A2 pre-mRNA because of a base substitution at the 5' splice donor site of intron 2 of the HLA-A2 gene. As a result, HLA-A2 transcripts are spliced to two aberrant forms, one with exon 2 skipping and the other with intron 2 retention. The latter is not translated because of an early premature stop codon in the retained intron. In contrast, the transcript with exon 2 skipping is translated to a truncated HLA-A2 heavy chain without the alpha(1) domain. Such a polypeptide is synthesized in vitro but is not detectable in cells, probably because of the low steady state level of the corresponding mRNA and the low translation efficiency. These results indicate that a single mutational event in an HLA class I gene is sufficient for loss of the corresponding allele. This may account, at least in part, for the high frequency of selective HLA class I allele loss in melanoma cells. Our conclusion emphasizes the need to implement active specific immunotherapy with a combination of peptides presented by various HLA class I alleles. This strategy may counteract the ability of melanoma cells with selective HLA class I allele loss to escape from immune recognition.

Identification of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART-1(27-35).

CTL reactivity to the epitope MART-1(27-35), of the melanoma (self) antigen MART-1/melan A is frequently observed in tumor-infiltrating lymphocytes and may be readily elicited from the peripheral blood of melanoma patients that express HLA-A*0201. Available data suggest that these observations contrast with those made for other HLA-A*0201-presented melanoma self antigens regarding the regularity of observed CTL responses. Based on preliminary findings, we hypothesized that the CTL response to MART-1 might be augmented in part by T cell encounters with peptides derived from sources other than MART-1, which show sequence similarity to MART-1(27-35). To test this idea, a protein database search for potential MART-1 epitope mimics was done using criteria developed from analyses of effector recognition of singly-substituted peptide analogues of MART-1(27-35). Synthetic peptides were made for a portion of the sequences retrieved; 12/40 peptides tested were able to sensitize target cells for lysis by one or more anti-MART-1 effectors. The peptides recognized correspond to sequences occurring in a variety of proteins of viral, bacterial, and human (self) origin. One peptide derives from glycoprotein C of the common pathogen HSV-1; cells infected with recombinant vaccinia virus encoding native glycoprotein C were lysed by anti-MART-1 effectors. Our results overall indicate that sequences conforming to the A2.1 binding motif and possessing features essential to recognition by anti-MART-1 CTL occur frequently in proteins. These findings further suggest that T cells might encounter a variety of such sequences in vivo, and that epitope mimicry may play a role in modulating the CTL response to MART-1(27-35).

Inherited polymorphisms in the RNA-mediated interference machinery affect microRNA expression and lung cancer survival.

BACKGROUND: MicroRNAs (miRs) have an important role in lung carcinogenesis and progression. Single-nucleotide polymorphisms (SNPs) in genes involved in miR biogenesis may affect miR expression in lung tissue and be associated with lung carcinogenesis and progression. METHODS: we analysed 12 SNPs in POLR2A, RNASEN and DICER1 genes in 1984 cases and 2073 controls from the Environment And Genetics in Lung cancer Etiology (EAGLE) study. We investigated miR expression profiles in 165 lung adenocarcinoma (AD) and 125 squamous cell carcinoma tissue samples from the same population. We used logistic and Cox regression models to examine the association of individual genotypes and haplotypes with lung cancer risk and with lung cancer-specific survival, respectively. SNPs-miR expression associations in cases were assessed using two-sample t-tests and global permutation tests. RESULTS: a haplotype in RNASEN (Drosha) was significantly associated with shorter lung cancer survival (hazard ratio=1.86, 95% CI=1.19-2.92, P=0.007). In AD cases, a SNP within the same haplotype was associated with reduced RNASEN mRNA expression (P=0.013) and with miR expression changes (global P=0.007) of miRs known to be associated with cancer (e.g., let-7 family, miR-21, miR-25, miR-126 and miR15a). CONCLUSION: inherited variation in the miR-processing machinery can affect miR expression levels and lung cancer-specific survival.

High-fidelity mRNA amplification for gene profiling.

The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.

Real-time quantitative polymerase chain reaction assessment of immune reactivity in melanoma patients after tumor peptide vaccination.

BACKGROUND: Monitoring the immune response to epitope-specific vaccination in cancer patients is important for vaccine development. The traditional method, in which the in vitro sensitization of peripheral blood mononuclear cells (PBMCs) with epitope is compared before and after vaccination, is time-consuming and allows only a qualitative assessment of the response. We used a rapid, quantitative, real-time polymerase chain reaction (PCR) assay to directly measure the immune reactivity of patients' PBMCs to the vaccine epitope. METHODS: PBMCs were obtained from melanoma patients before and after two rounds of vaccination with either g209-2M, a peptide derived from melanoma protein gp100 (n = 24), or ESg209-2M, a modified version of this peptide (n = 20). PBMCs were tested for immune reactivity by assaying interferon gamma (IFN gamma) protein release after in vitro sensitization with the epitope or for IFN gamma messenger RNA expression by real-time PCR. A twofold or more increase in IFN gamma protein release or a 1.5-fold or more increase in IFN gamma transcript accumulation in PBMCs after vaccination was considered to be evidence of a specific response. Correlation between the two methods was tested by use of the Spearman correlation coefficient after the results were ranked as positive or negative. All statistical tests were two-sided. RESULTS: The results obtained with the two methods were strongly correlated (Spearman's rho = 0.72; P =.0006). The g209-2M and Esg209-2M peptides resulted in similar percentages of vaccine-specific reactivity in PBMCs after in vitro sensitization (63% and 65% of patients, respectively; Fisher's exact test P =.6 for comparison of the two groups). The PCR method could detect vaccine-specific reactivity in a subset of patients (38% and 35% of patients, respectively; Fisher's exact test P =.7 for comparison of the two groups). CONCLUSION: Vaccination induces circulating antitumor lymphocytes, albeit in low frequencies, capable of directly reacting with tumor antigen. PBMCs of vaccinated individuals can respond to a vaccine-specific stimulus in a direct assay that does not require prolonged in vitro manipulations.

Loss of functional beta 2-microglobulin in metastatic melanomas from five patients receiving immunotherapy.

BACKGROUND: In a subset of patients with metastatic melanoma, T lymphocytes bearing the cell-surface marker CD8 (CD8+ T cells) can cause the regression of even large tumors. These antitumor CD8+ T cells recognize peptide antigens presented on the surface of tumor cells by major histocompatibility complex (MHC) class I molecules. The MHC class I molecule is a heterodimer composed of an integral membrane glycoprotein designated the alpha chain and a noncovalently associated, soluble protein called beta2-microglobulin (beta 2m). Loss of beta 2m generally eliminates antigen recognition by antitumor CD8+ T cells. PURPOSE: We studied the loss of beta 2m as a potential means of tumor escape from immune recognition in a cohort of patients receiving immunotherapy. METHODS: We successfully grew 13 independent tumor cell cultures from tumor specimens obtained from 13 patients in a cohort of 40 consecutive patients undergoing immunotherapy for metastatic melanoma and for whom tumor specimens were available. These cell lines, as well as another melanoma cell line (called 1074mel) that had been derived from tumor obtained from a patient in a cytokine-gene therapy study, were characterized in vitro cytofluorometrically for MHC class I expression and by northern and western blot analyses for messenger RNA (mRNA) and protein expression, respectively, and ex vivo by immunohistochemistry. RESULTS: After one melanoma cell line (1074mel) was found not to express functional beta 2m by cytofluorometric analysis, four (31%) of the 13 newly established melanoma cell lines were found to have an absolute lack of functional MHC class I expression. Northern blot analysis of RNA extracted from the five cell lines exhibiting no functional MHC class I expression showed that these cells contained normal levels of alpha-chain mRNA but variable levels of beta 2m mRNA. In addition, no immunoreactive beta 2m protein was detected by western blot analysis. When human beta 2m was transiently expressed with the use of a recombinant vaccinia virus, cell-surface MHC class I expression was reconstituted and the ability of these five cell lines to present endogenous antigens was restored. Immunohistochemical staining of tumor sections revealed a lack of immunoreactive MHC class I in vivo, supporting the notion that the in vitro observations were not artifactual. Furthermore, archival tumor sections obtained from patients prior to immunotherapy were available from three patients and were found to be beta 2m positive. This result was consistent with the hypothesis that loss of beta 2m resulted from immunotherapy. CONCLUSIONS: These data suggest that the loss of beta 2m may be a mechanism whereby tumor cells can acquire immunoresistance. This study represents the first characterization of a molecular route of escape of tumors from immune recognition in a cohort of patients being treated with immunotherapy.

Immunizing patients with metastatic melanoma using recombinant adenoviruses encoding MART-1 or gp100 melanoma antigens.

BACKGROUND: The characterization of the genes encoding melanoma-associated antigens MART-1 or gp100, recognized by T cells, has opened new possibilities for the development of immunization strategies for patients with metastatic melanoma. With the use of recombinant adenoviruses expressing either MART-1 or gp100 to immunize patients with metastatic melanoma, we evaluated the safety, immunologic, and potential therapeutic aspects of these immunizations. METHODS: In phase I studies, 54 patients received escalating doses (between 10(7) and 10(11) plaque-forming units) of recombinant adenovirus encoding either MART-1 or gp100 melanoma antigen administered either alone or followed by the administration of interleukin 2 (IL-2). The immunologic impact of these immunizations on the development of cellular and antibody reactivity was assayed. RESULTS: Recombinant adenoviruses expressing MART-1 or gp100 were safely administered. One of 16 patients with metastatic melanoma receiving the recombinant adenovirus MART-1 alone experienced a complete response. Other patients achieved objective responses, but they had received IL-2 along with an adenovirus, and their responses could be attributed to the cytokine. Immunologic assays showed no consistent immunization to the MART-1 or gp100 transgenes expressed by the recombinant adenoviruses. High levels of neutralizing antibody were found in the pretreatment sera of the patients. CONCLUSIONS: High doses of recombinant adenoviruses could be safely administered to cancer patients. High levels of neutralizing antibody present in patients' sera prior to treatment may have impaired the ability of these viruses to immunize patients against melanoma antigens.

Immunization against epitopes in the human melanoma antigen gp100 following patient immunization with synthetic peptides.

gp1OO is a melanocytic lineage-specific antigen recognized by tumor-infiltrating lymphocytes, the adoptive transfer of which is associated with tumor regression in melanoma patients. In this study, peripheral blood mononuclear cells (PBMCs) were harvested from HLA-A2+ melanoma patients before and after immunization with G9-209 (ITDQVPFSY), G9-280 (YLEPGPVTA), or G9-154 (KTWGQYWQV) peptides in Incomplete Freund's Adjuvant and were tested for the ability to be sensitized in vitro using PBMCs pulsed with the native peptides. In addition, PBMCs from patients receiving the G9-209 or G9-280 peptide were stimulated in vitro with peptides modified at anchor residues to enhance binding to HLA-A2: G9-209/2M (IMDQVPFSY) or G9-280-9V (YLEPGPVTV). In patients immunized with G9-209, a single in vitro restimulation with G9-209/2M resulted in the generation of specific antipeptide lymphocytes from seven of seven postimmune PBMCs and only three of seven preimmune PBMCs. In patients immunized with G9-280, a single in vitro restimulation with G9-280/9V resulted in the generation of specific antipeptide lymphocytes from five of six postimmune PBMCs and four of six preimmune PBMCs. In almost all cases, CTLs raised against modified epitopes were capable of recognizing targets displaying the native nonamers. Several anti-G9-209 and anti-G9-209/2M CTLs also demonstrated specific lysis of, and specific IFN-gamma release in response to, gp1OO+-established cell lines. Thus, using peptides modified to enhance immunogenicity for in vitro stimulation improved the sensitivity of immune monitoring of patients immunized with synthetic peptides. These results demonstrate that immunization with a peptide derived from a tumor-associated protein such as gp100 can provoke a measurable antitumor immune response in cancer patients.

Recognition of multiple epitopes in the human melanoma antigen gp100 by peripheral blood lymphocytes stimulated in vitro with synthetic peptides.

gp100 is a melanocyte lineage-specific antigen recognized by tumor-infiltrating lymphocytes whose adoptive transfer has been associated with tumor regression in patients with metastatic melanoma. The peripheral blood mononuclear cells of five melanoma patients were sensitized in vitro with synthetic peptides to elicit antigen-specific cytotoxic T lymphocyte (CTL) lines against four gp100 epitopes. These epitope-specific CTL lines were generated following weekly in vitro stimulation with the synthetic decamer G10(476) (V-L-Y-R-Y-G-S-F-S-V) or the nonamers G9(280) (Y-L-E-P-G-P-V-T-A), G9(154) (K-T-W-G-Q-Y-W-Q-V), or G9(209) (I-T-D-Q-V-P-F-S-V) pulsed onto autologous irradiated peripheral blood mononuclear cells. These lines grew as long as 4 months in culture in low-dose interleukin 2 (30 IU/ml) and exhibited antigen-specific, MHC class I-restricted lysis of peptide-pulsed T2 cells and HLA-A2+, gp100+ established melanoma cell lines. G10(476)- and G9(280)-specific CTLs demonstrated specific release of granulocyte-macrophage-colony-stimulating factor and tumor necrosis factor alpha in response to T2 cells pulsed with relevant peptide, as well as to gp100+ melanoma cell lines. These results demonstrate that several peptides derived from the gp100 protein are presented on the surface of melanoma cells and are sufficiently immunogenic to generate, in vitro, potent CTLs capable of cytolysis and the secretion of cytokines. Therefore, for HLA-A2+ melanoma patients, these and possibly other gp100 peptides could represent good candidates for antigen-specific immunotherapy either singly or in a multivalent regimen.

Induction of specific CD8+ T-lymphocyte responses using a human papillomavirus-16 E6/E7 fusion protein and autologous dendritic cells.

When intracellular viral proteins are degraded, only a limited number of peptide epitopes are capable of eliciting specific CD8+ cellular immune responses for a given human leukocyte antigen (HLA) haplotype. We sought to induce CD8+ T-lymphocyte (CTL) responses to human papillomavirus-16 (HPV-16) E6 and E7 proteins using a recombinant E6/E7 fusion protein and autologous human dendritic cells (DCs). CTLs were generated by in vitro stimulation using a recombinant HPV-16 E6/E7 fusion protein and autologous DCs from a healthy HLA-A*0201 donor. CTL specificity was assessed by cytokine release assays when the cells were reacted with autologous DC targets coincubated with the E6/E7 fusion protein. These CTLs were also reacted with the immunodominant E7 peptides (E711-20 and E7(86-93)) and DCs as a target. As a negative control, DCs were incubated with or without an irrelevant control protein (Helicobacter pylori) as target for the E6/E7-induced CTLs. The E6/E7-induced CTLs were capable of specific recognition of target DCs coincubated with E6/E7 but not the control protein. When E6/E7-specific CTLs were reacted with DCs and either E7(11-20) or E7(86-93), specific peptide recognition was also detected. These data demonstrate that specific CTLs can be elicited using autologous human DCs and a HPV-16 E6/E7 fusion protein. Therefore, extracellular viral proteins seem to be engulfed and processed by DCs; then the immunodominant HLA-A2-restricted peptides become available for CD8+ T-lymphocyte recognition. These data suggest that vaccine strategies using recombinant viral proteins may overcome the limitation of peptide epitopes for specific HLA haplotypes and may, therefore, permit more generalized clinical application.

HLA association with response and toxicity in melanoma patients treated with interleukin 2-based immunotherapy.

Peripheral blood lymphocytes from 146 patients with metastatic melanoma undergoing interleukin 2 (IL-2)-based immunotherapy were characterized for HLA A, B, Cw, DR, DQw, and DRw specificities. Patients had been enrolled into sequential treatment protocols with either IL-2 alone (28) or in combination with tumor-infiltrating lymphocytes (TILs) (86), alpha-interferon (26), lymphokine-activated killer cells (16), radiation therapy (7), cyclophosphamide (3), tumor necrosis factor (1), and interleukin 4 (1) for a total of 168 courses of therapy. HLA phenotype was then correlated with response rate and toxicity to IL-2. We noted: (a) a significant difference in the frequency of A11 (20.5% versus 10.2%; P < 0.05) allele between melanoma patients and the North American Caucasian population; (b) a significantly higher frequency of A11 phenotype among responders (40.5%) than in the melanoma patient population (20.5%; P < 0.01), which was even more obvious among patients responding to TIL therapy (47.4% versus 22.1%; P < 0.05); within TIL patients, responders also had an increased frequency of A19 (42.1% versus 25.6%; P < 0.05); (c) a correlation between the number of TILs received and response rate (P < 0.005); and (d) an association between DR4 haplotype and decreased tolerance to IL-2 among the patients receiving TILs (P = 0.01). These results suggest that, in melanoma patients, some HLA Class I specificities may predict for a greater likelihood of response to IL-2-based therapy, while HLA Class II phenotype correlates with tolerance to the combination of TIL and IL-2 therapy.

Antigenicity of fusion proteins from sarcoma-associated chromosomal translocations.

Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Specific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunological relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously expressing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the development of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations.

Host response to cytomegalovirus infection as a determinant of susceptibility to coronary artery disease: sex-based differences in inflammation and type of immune response.

BACKGROUND: Positive and negative associations between cytomegalovirus (CMV) infection and coronary artery disease (CAD) have been reported. We postulated that the susceptibility to CMV-induced CAD might relate to patterns of inflammatory and immune responses to CMV infection and that sex might have an effect on these responses. METHODS AND RESULTS: In 151 men and 87 women being evaluated for CAD, blood samples were tested for humoral (Ab+) and cellular (Tc+) responses to CMV and for C-reactive protein (CRP). In men, an elevated CRP level was a significant determinant of CAD even after adjustment for CAD risk factors (OR, 3.1; 95% CI, 1.21 to 7. 97). CMV seropositivity was associated with elevated CRP levels on multivariate analysis (P:=0.006). In contrast, in women, CMV seropositivity was independently predictive of CAD (OR, 41.8; 95% CI, 4.12 to 423.74). CRP level in women with CAD was >25% higher than those without CAD, but the difference did not reach statistical significance. Importantly, compared with CMV Ab-/Tc- women, CAD prevalence was higher in Ab+/Tc- and Ab+/Tc+ (13% versus 68% and 64%, both P:<0.005) but not in Ab-/Tc+ women (25%). There were no differences in age, smoking, diabetes, hypertension, and hypercholesterolemia among women with different types of immune responses to CMV infection. CONCLUSIONS: The mechanisms by which CMV predisposes to CAD in men and women may be different. In men, CMV appears to contribute to CAD risk, insofar as it predisposes to inflammation. In women, other mechanisms, possibly related to the type of immune response generated by the host, appear to be responsible for the proatherogenic effects of CMV.

Combination therapy with interferon alfa-2a and interleukin-2 for the treatment of metastatic cancer.

PURPOSE: Here we report the long-term follow-up evaluation of a phase I/II study of toxicity and response of combination interferon alfa-2a (IFN alpha) and interleukin-2 (IL-2) in patients with metastatic cancer. PATIENTS AND METHODS: From November 1987 through October 1990, 189 patients were treated with 379 courses. IFN alpha (3 x 10(6) U/m2) was administered three times per day as an intravenous (IV) bolus with IV IL-2 2.6 x 10(6) IU/m2 (six patients, group 1), 7.8 x 10(6) IU/m2 (32 patients, group 2), or 11.7 x 10(6) IU/m2 (26 patients, group 3). Subsequently, IFN alpha dose was escalated to 6 x 10(6) U/m2 plus IL-2 11.7 x 10(6) IU/m2 (22 patients, group 4). Two further dosage schedules of IL-2 were tested at 7.8 x 10(6) IU/m2 (29 patients, group 5) and 15.6 x 10(6) IU/m2 (74 patients, group 6); however, because of IFN alpha-related toxicity, these two groups received IFN alpha once per day (6 x 10(6) U/m2). A treatment course consisted of two cycles (maximum, 15 doses per cycle) separated by a 10-day interval. RESULTS: All patients were assessable for response: 82 patients had melanoma, 75 renal cell carcinoma (RCC), and 16 colorectal cancer. There were two treatment-related deaths. The objective response rate was 23% (43 patients). Response rates were 17%, 19%, 19%, 32%, 41%, and 16%, respectively, for groups 1 through 6. Ten responses are still ongoing (nine in RCC patients) at 57 to 74 months, and 21 patients are alive, for an overall 5-year survival rate of 11%. The median potential follow-up period was 65 months. Although a significantly higher response rate was noted for group 4 (highest dose of IFN alpha three times per day), no benefit for survival and increased toxicity were noted in this group. CONCLUSION: Based on these findings, we conclude that further studies of this combination treatment are not warranted.