Quality of life assessment in diffuse large B-cell lymphoma (DLBCL) in REFLECT: a prospective, non-interventional, multicenter, German study, assessing Sandoz rituximab in combination with CHOP.

Health-related quality of life (HRQoL) data are important indicators of health status in patients with lymphoma. The objective of this analysis was to assess the impact of treatment with Sandoz rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) on HRQoL in treatment-naïve adult patients with diffuse large B-cell lymphoma (DLBCL) included in the prospective, real-world REFLECT study. REFLECT is the first prospective study to assess HRQoL in patients with DLBCL treated with a rituximab biosimilar. HRQoL was assessed via the patient-reported European Organization for Research and Treatment of Cancer Core Quality of Life questionnaire at baseline, mid-treatment (month 3), end of treatment (month 6), and follow-up (months 9 and 12). Subgroup analyses were performed to evaluate the influence of baseline characteristics on HRQoL, and associations between baseline HRQoL and treatment response. HRQoL was assessed in 169 patients. Mean global health status score remained stable from baseline (54.8) to mid-treatment (month 3; 54.7), before steadily improving through to end of treatment (month 6; 61.4), and follow-up month 9 (64.9) and month 12 (68.8). Similar trends were observed across most functional and symptom subscales. Higher cognitive, physical, or role functioning, and less appetite loss, diarrhea, fatigue, or pain at baseline, were all associated with an improved likelihood of reaching a complete versus partial response at the end of treatment. Overall, these findings confirm the HRQoL benefits of R-CHOP therapy in treatment-naïve adult patients with DLBCL, and suggest that baseline HRQoL may be predictive of treatment response.

Nivolumab and ipilimumab in recurrent or refractory cancer of unknown primary: a phase II trial.

Cancer of unknown primary has a dismal prognosis, especially following failure of platinum-based chemotherapy. 10-20% of patients have a high tumor mutational burden (TMB), which predicts response to immunotherapy in many cancer types. In this prospective, non-randomized, open-label, multicenter Phase II trial (EudraCT 2018-004562-33; NCT04131621), patients relapsed or refractory after platinum-based chemotherapy received nivolumab and ipilimumab following TMBhigh vs. TMBlow stratification. Progression-free survival (PFS) represented the primary endpoint; overall survival (OS), response rates, duration of clinical benefit and safety were the secondary endpoints. The trial was prematurely terminated in March 2021 before reaching the preplanned sample size (n = 194). Among 31 evaluable patients, 16% had a high TMB ( > 12 mutations/Mb). Overall response rate was 16% (95% CI 6-34%), with 7.7% (95% CI 1-25%) vs. 60% (95% CI 15-95%) in TMBlow and TMBhigh, respectively. Although the primary endpoint was not met, high TMB was associated with better median PFS (18.3 vs. 2.4 months) and OS (18.3 vs. 3.6 months). Severe immune-related adverse events were reported in 29% of cases. Assessing on-treatment dynamics of circulating tumor DNA using combined targeted hotspot mutation and shallow whole genome sequencing as part of a predefined exploratory analysis identified patients benefiting from immunotherapy irrespective of initial radiologic response.

REFLECT: prospective multicenter non-interventional study evaluating the effectiveness and safety of Sandoz rituximab (SDZ-RTX; Rixathon®) in combination with CHOP for the treatment of patients with previously untreated CD20-positive diffuse large B-cell lymphoma.

Background: REFLECT is the first prospective study of Sandoz biosimilar rituximab (SDZ-RTX) in patients with diffuse large B-cell lymphoma (DLBCL). Objective: To evaluate the 2-year effectiveness and safety of SDZ-RTX as first-line treatment for DLBCL. Design: Real-world, multicenter, open-label, single-arm, non-interventional, post-approval study of SDZ-RTX in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in patients with treatment-naïve CD20­positive DLBCL. Methods: Treatment-naïve, CD20-positive adult patients (⩾18 years) with DLBCL eligible for therapy with R-CHOP were treated with SDZ-RTX-CHOP every 2 or 3 weeks for 6-8 cycles. The effectiveness of SDZ-RTX was measured by the complete response (CR) rate at the end of R-CHOP treatment, as assessed by the treating physician. Progression-free survival (PFS) was assessed at 24 months. Results: A total of 169 patients [52.1% female, median (range) age 70 (24-94) years] with DLBCL were included in the full analysis set. At baseline, 19.5% and 24.3% of patients had Ann Arbor disease stage III or IV, respectively, and most patients (80.5%) had Eastern Cooperative Oncology Group Performance Status of 0 or 1. A total of 100 (59.2%) patients completed the 24-month observation period. In total, 110 [65.1%; 95% confidence interval (CI): 57.4-72.3] patients achieved CR as best response and 50 (29.6%; 95% CI: 22.8-37.1) patients achieved partial response. Overall best response rate was 94.7% (95% CI: 90.1-97.5). One-year PFS was 84.9% (95% CI: 78.2-89.6), while 2-year PFS was 78.5% (95% CI: 70.9-84.4); median PFS was not reached within the observational period. A total of 143 (84.6%) patients experienced ⩾1 adverse event, 53 (31.4%) of which were suspected to be related to study drug. Conclusion: This real-world, 2-year study reconfirms that first-line treatment of CD20-positive DLBCL with R-CHOP using SDZ-RTX is effective and well tolerated. Registration: N/A.

REFLECT: A study evaluating Sandoz biosimilar rituximab (Rixathon ^® ) in combination with CHOP for the treatment of patients with previously untreated diffuse large B-cell lymphoma Why was this study done? • Biosimilars are biologic medicines that are highly similar to a reference biologic medicine that is already approved and has been used in patients for several years. • The REFLECT study was the first study of a biosimilar medicine (Sandoz biosimilar rituximab) in patients with a type of lymphatic cancer called diffuse large B-cell lymphoma (DLBCL). What did the researchers do? • Sandoz biosimilar rituximab was given as part of the standard treatment (cyclophosphamide, doxorubicin, vincristine, and prednisone; R-CHOP) in patients with DLBCL who had not received treatment before. • The researchers aimed to evaluate how well Sandoz biosimilar rituximab worked over a 2-year period. • The researchers also aimed to look at the safety of Sandoz biosimilar rituximab. • Patients with DLBCL had to be ⩾18 years of age, in need of treatment, and were classed as suitable for treatment with R-CHOP by their doctor. • Patients were treated with R-CHOP including Sandoz biosimilar rituximab every 2 or 3 weeks for 6­8 cycles. What did the researchers find? • A total of 169 patients with DLBCL were included in the study. • Just over half (52%) were female and the average age was 67 years. • Nearly 6 out of 10 (59%) patients completed the 2-year study. • More than 6 out of 10 (65%) patients achieved complete response and 3 out of 10 (30%) achieved partial response. • The overall response rate was 95%. • One-year progression-free survival was 85%, and 2-year progression-free survival was 79%. • Regarding safety, 85% of patients experienced at least one adverse event; just over 3 out of 10 (31%) of these were suspected to be related to the study drug. What do the findings mean? • This 2-year study shows that R-CHOP including Sandoz biosimilar rituximab is effective and well tolerated as the first treatment given to patients with DLBCL.

B-cell receptors of EBV-negative Burkitt lymphoma bind modified isoforms of autoantigens.

Burkitt lymphoma (BL) represents the most aggressive B-cell-lymphoma. Beside the hallmark of IG-MYC-translocation, surface B-cell receptor (BCR) is expressed, and mutations in the BCR pathway are frequent. Coincidental infections in endemic BL, and specific extra-nodal sites suggest antigenic triggers. To explore this hypothesis, BCRs of BL cell lines and cases were screened for reactivities against a panel of bacterial lysates, lysates of Plasmodium falciparum, a custom-made virome array and against self-antigens, including post-translationally modified antigens. An atypically modified, SUMOylated isoform of Bystin, that is, SUMO1-BYSL was identified as the antigen of the BCR of cell line CA46. SUMO1-BYSL was exclusively expressed in CA46 cells with K139 as site of the SUMOylation. Secondly, an atypically acetylated isoform of HSP40 was identified as the antigen of the BCR of cell line BL41. K104 and K179 were the sites of immunogenic acetylation, and the acetylated HSP40 isoform was solely present in BL41 cells. Functionally, addition of SUMO1-BYSL and acetylated HSP40 induced BCR pathway activation in CA46 and BL41 cells, respectively. Accordingly, SUMO1-BYSL-ETA' immunotoxin, produced by a two-step intein-based conjugation, led to the specific killing of CA46 cells. Autoantibodies directed against SUMO1-BYSL were found in 3 of 14 (21.4%), and autoantibodies against acetylated HSP40 in 1/14(7.1%) patients with sporadic Burkitt-lymphoma. No reactivities against antigens of the infectious agent spectrum could be observed. These results indicate a pathogenic role of autoreactivity evoked by immunogenic post-translational modifications in a subgroup of sporadic BL including two EBV-negative BL cell lines.

Identification of the atypically modified autoantigen Ars2 as the target of B-cell receptors from activated B-cell-type diffuse large B-cell lymphoma.

It has been suggested that B-cell receptor (BCRs) stimulation by specific antigens plays a pathogenic role in diffuse large B-cell lymphoma (DLBCL). Here, it was the aim to screen for specific reactivities of DLBCL-BCRs in the spectrum of autoantigens and antigens of infectious origin. Arsenite resistance protein 2 (Ars2) was identified as the BCR target of 3/5 ABC-type DLBCL cell lines and 2/11 primary DLBCL cases. Compared to controls, Ars2 was hypo-phosphorylated exclusively in cases and cell lines with Ars2-specific BCRs. In a validation cohort, hypo-phosphorylated Ars2 was found in 8/31 ABC-type, but only 1/20 germinal center B cell (GBC)-like type DLBCL. Incubation with Ars2 induced BCR-pathway activation and increased proliferation, while an Ars2/ETA-toxin conjugate induced killing of cell lines with Ars2-reactive BCRs. Ars2 appears to play a role in a subgroup of ABC-type DLBCLs. Moreover, transformed DLBCL lines with Ars2-reactive BCRs still show growth advantage after incubation with Ars2. These results provide knowledge about the pathogenic role of a specific antigen stimulating the BCR pathway in DLCBL.

KIT D816 mutated/CBF-negative acute myeloid leukemia: a poor-risk subtype associated with systemic mastocytosis.

KIT D816 mutations (KIT D816mut) are strongly associated with systemic mastocytosis (SM) but are also detectable in acute myeloid leukemia (AML), where they represent an adverse prognostic factor in combination with core binding factor (CBF) fusion genes. Here, we evaluated the clinical and molecular features of KIT D816mut/CBF-negative (CBFneg) AML, a previously uncharacterized combination. All KIT D816mut/CBFneg cases (n = 40) had histologically proven SM with associated AML (SM-AML). Molecular analyses revealed at least one additional somatic mutation (median, n = 3) beside KIT D816 (e.g., SRSF2, 38%; ASXL1, 31%; RUNX1, 34%) in 32/32 (100%) patients. Secondary AML evolved in 29/40 (73%) patients from SM ± associated myeloid neoplasm. Longitudinal molecular and cytogenetic analyses revealed the acquisition of new mutations and/or karyotype evolution in 15/16 (94%) patients at the time of SM-AML. Median overall survival (OS) was 5.4 months. A screen of two independent AML databases (AMLdatabases) revealed remarkable similarities between KIT D816mut/CBFneg SM-AML and KIT D816mut/CBFneg AMLdatabases (n = 69) with regard to KIT D816mut variant allele frequency, mutation profile, aberrant karyotype, and OS suggesting underlying SM in a significant proportion of AMLdatabases patients. Bone marrow histology and reclassification as SM-AML has important clinical implications regarding prognosis and potential inclusion of KIT inhibitors in treatment concepts.

Learning from the failures of drug discovery in B-cell non-Hodgkin lymphomas and perspectives for the future: chronic lymphocytic leukemia and diffuse large B-cell lymphoma as two ends of a spectrum in drug development.

INTRODUCTION: Despite substantial recent advances, there is still an unmet need for better therapies in B-cell non Hodgkin lymphomas (B-NHL), especially in relapsed or refractory disease. Many novel targeted drugs have been developed based on a better molecular understanding of B-NHL. Areas covered: This article focuses on chronic lymphocytic leukemia (CLL) as a representative for indolent lymphomas and paradigmatic for the tremendous progress in treating B-NHL on the one hand and diffuse large B-cell lymphoma (DLBCL) as a representative for aggressive lymphomas and paradigmatic for many unsolved problems in lymphoma treatment or the other hand. We highlight salient points in current therapies targeting genetic, epigenetic, immunological and microenvironmental alterations. Possible reasons for drug failure in clinical trials like tumor heterogeneity, clonal evolution and drug resistance mechanisms are discussed. Based thereon, some perspectives for further drug discovery are given. Expert opinion: In view of the pathogenetic complexity of lymphomas, therapies targeting exclusively a single alteration may fail because resistance mechanisms are present either initially or evolve during treatment. Therefore, future therapies in B-NHL may have to target the greatest possible number of genetic, immunological or epigenetic alterations still allowing tolerability and to monitor these alterations during therapy.

Autoantibodies against lamin C, NA14 and CK15 in primary vasculitides or autoimmune diseases with secondary vasculitis.

OBJECTIVES: Autoantibodies may play a role in the pathogenesis of primary vasculitides (PVs) like giant cell arteritis (GCA). We recently identified 3 cytoskeletal proteins (lamin C [laC], nuclear autoantigen of 14kD [NA14] and cytokeratin 15 [CK15]) as autoantigens in GCA and postulated a frequent autoantibody response against these antigens in PVs. METHODS: To test this hypothesis we studied a cohort of patients with PVs (n=61) and healthy individuals (n=27) for the presence of these autoantibodies using a recombinant cDNA expression library. To define their specifity for PV, we also examined patients with other autoimmune diseases such as rheumatoid arthritis (RA) and connective tissue diseases (CTD). RESULTS: We found no statistically significant differences in autoantibody responses between patients with PV and healthy controls, although there was a trend for an association between PVs and the occurrence of antibodies against laC and CK15. However, in patients with RA (n=33) or Sjögren's syndrome (SS, n=11) with vasculitides we observed more frequently autoantibodies against NA14, laC and CK15 compared to healthy controls. In patients with systemic lupus erythematosus (SLE, n=23) autoantibodies against laC were more frequent. The presence of autoantibodies in RA, SS and SLE was associated with systemic secondary vasculitis (SSV). In RA, laC- and NA14-seropositive patients were in a more advanced disease stage than seronegative patients with more frequent extraarticular manifestations (p=0.004). In SLE laC-autoantibody-positive patients had a higher SLE activity index (p<0.001). CONCLUSIONS: Serum autoantibodies against laC and NA14 are frequent in patients with RA and CTD and are associated with extensive disease and SSV. The potential pathogenic and prognostic role of these antibodies should be further investigated.

The molecular basis for development of proinflammatory autoantibodies to progranulin.

Recently we identified in a wide spectrum of autoimmune diseases frequently occurring proinflammatory autoantibodies directed against progranulin, a direct inhibitor of TNFR1 & 2 and of DR3. In the present study we investigated the mechanisms for the breakdown of self-tolerance against progranulin. Isoelectric focusing identified a second, differentially electrically charged progranulin isoform exclusively present in progranulin-antibody-positive patients. Alkaline phosphatase treatment revealed this additional progranulin isoform to be hyperphosphorylated. Subsequently Ser81, which is located within the epitope region of progranulin-antibodies, was identified as hyperphosphorylated serine residue by site directed mutagenesis of candidate phosphorylation sites. Hyperphosphorylated progranulin was detected exclusively in progranulin-antibody-positive patients during the courses of their diseases. The occurrence of hyperphosphorylated progranulin preceded seroconversions of progranulin-antibodies, indicating adaptive immune response. Utilizing panels of kinase and phosphatase inhibitors, PKCß1 was identified as the relevant kinase and PP1 as the relevant phosphatase for phosphorylation and dephosphorylation of Ser81. In contrast to normal progranulin, hyperphosphorylated progranulin interacted exclusively with inactivated (pThr320) PP1, suggesting inactivated PP1 to cause the detectable occurrence of phosphorylated Ser81 PGRN. Investigation of possible functional alterations of PGRN due to Ser81 phosphorylation revealed, that hyperphosphorylation prevents the interaction and thus direct inhibition of TNFR1, TNFR2 and DR3, representing an additional direct proinflammatory effect. Finally phosphorylation of Ser81 PGRN alters the conversion pattern of PGRN. In conclusion, inactivated PP1 induces hyperphosphorylation of progranulin in a wide spectrum of autoimmune diseases. This hyperphosphorylation prevents direct inhibition of TNFR1, TNFR2 and DR3 by PGRN, alters the conversion of PGRN, and is strongly associated with the occurrence of neutralizing, proinflammatory PGRN-antibodies, indicating immunogenicity of this alternative secondary modification.

Management of diffuse large B-cell lymphoma (DLBCL).

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. While CHOP was the standard combination chemotherapy for 25 years, the incorporation of the CD20 antibody rituximab at the beginning of this century has considerably improved the outcome of all patients with DLBCL: Depending on the prognostic subgroup, only half to one-third of the patients die of their DLBCL compared to pre-rituximab era. Treatment is usually tailored according to the individual risk profile of a DLBCL patient according to the International Prognostic Index (IPI). Assignment of DLBCL according to the gene expression profile into DLBLC originating from a germinal center B cell (GC type) or from an activated B cell (ABC type) has provided novel insights into the pathogenesis of the respective DLBCL, identified molecules which are indispensable for the survival of the lymphoma cells and provided targets for novel "targeted therapies" drugs. Incorporating these new drugs into combination immunochemotherapy or substituting single drugs in the R-CHOP combination will result in even higher cure rates of and/or less toxicity for patients with DLBCL in the decade to come.

CD4⁺ T cells in chronic autoantigenic stimulation in MGUS, multiple myeloma and Waldenström's macroglobulinemia.

Hyperphosphorylated paratarg-7 (pP-7) carrier state is the strongest and most frequent molecular risk factor for MGUS, multiple myeloma (MM) and Waldenström's macroglobulinemia (WM), inherited autosomal-dominantly and, depending on the ethnic background, found in up to one third of patients with MGUS/MM. Since P-7 is the antigenic target of paraproteins that do not distinguish between wtP-7 and pP-7, we investigated CD4(+) T-cell responses in pP-7(+) patients and controls. Peptides spanning amino acids 1-35 or 4-31 containing phosphorylated or nonphosphorylated serine17 were used for stimulation. CD4(+) cells from 9/14 patients (65%) showed a pP-7 specific HLA-DR restricted response. These results demonstrate that pP-7 specific CD4(+) cells can mediate help for pP-7 specific chronic antigenic stimulation of P-7 specific B cells, which might ultimately result in the clonal evolution of a B cell into MGUS/MM/WM producing a P-7 specific paraprotein. Prerequisites for pP-7 specific stimulation of CD4(+) cells appear to be both a pP-7 carrier state and an HLA-DR subtype able to present and recognize pP-7. Our results serve as an explanation for the exclusive autoimmunogenicity of the hyperphosphorylated variant of P-7 and for the different hazard ratios of pP-7 carriers from different ethnic origins to develop MGUS/MM/WM.

Sumoylated HSP90 is a dominantly inherited plasma cell dyscrasias risk factor.

Posttranslationally modified proteins serve as autoimmunogenic targets in a wide spectrum of autoimmune diseases. Here, we identified a posttranslationally modified paraprotein target (paratargs) in monoclonal gammopathies of undetermined significance (MGUS), multiple myelomas (MM), and Waldenstrom's macroglobulinemias (WM) using protein macroarrays that were sumoylated and screened for reactivity with paraproteins from MGUS, MM, and WM patients. We found that paraproteins from a proportion of European, African-American, and Japanese patients specifically reacted with the sumoylated heat-shock protein 90 ß isoform-α (HSP90-SUMO1, where SUMO indicates small ubiquitin-like modifier), while no reactivity with HSP90-SUMO1 was detected in over 800 controls. HSP90-SUMO1 was present in blood cells from all patients with HSP90-SUMO1-binding paraproteins. We determined that the HSP90-SUMO1 carrier state is autosomal-dominantly inherited and caused by the inability of SUMO peptidase sentrin/SUMO-specific protease 2 (SENP2) to desumoylate HSP90-SUMO1. HSP90-SUMO1 was detected in a small percentage of healthy individuals from all backgrounds; however, only MGUS, MM, and WM patients who were HSP90-SUMO1 carriers produced HSP90-SUMO1-specific paraproteins, suggesting that sumoylated HSP90 promotes pathogenesis of these diseases through chronic antigenic stimulation. This study demonstrates that harboring HSP90-SUMO1 identifies healthy individuals at risk for plasma cell dyscrasias and that dominant inheritance of posttranslationally modified autoantigenic paratargs is one of the strongest molecular defined risk factors for MGUS, MM, and WM.

EBV-transformed lymphoblastoid cell lines as vaccines against cancer testis antigen-positive tumors.

EBV-transformed lymphoblastoid cell lines (LCL) are potent antigen-presenting cells. To investigate their potential use as cancer testis antigen (CTA) vaccines, we studied the expression of 12 cancer testis (CT) genes in 20 LCL by RT-PCR. The most frequently expressed CT genes were SSX4 (50 %), followed by GAGE (45 %), SSX1 (40 %), MAGE-A3 and SSX2 (25 %), SCP1, HOM-TES-85, MAGE-C1, and MAGE-C2 (15 %). NY-ESO-1 and MAGE-A4 were found in 1/20 LCL and BORIS was not detected at all. Fifteen of 20 LCL expressed at least one antigen, 9 LCL expressed ≥2 CT genes, and 7 of the 20 LCL expressed ≥4 CT genes. The expression of CT genes did not correlate with the length of in vitro culture, telomerase activity, aneuploidy, or proliferation state. While spontaneous expression of CT genes determined by real-time PCR and Western blot was rather weak in most LCL, treatment with DNA methyltransferase 1 inhibitor alone or in combination with histone deacetylase inhibitors increased CTA expression considerably thus enabling LCL to induce CTA-specific T cell responses. The stability of the CT gene expression over prolonged culture periods makes LCL attractive candidates for CT vaccines both in hematological neoplasias and solid tumors.

Mutated Ras-transfected, EBV-transformed lymphoblastoid cell lines as a model tumor vaccine for boosting T-cell responses against pancreatic cancer: a pilot trial.

Genetically modified lymphoblastoid cell lines (LCL) have been shown to be an attractive alternative source of antigen-presenting cells for cancer vaccination in vitro. We tested their application in patients with pancreatic cancer in a phase I clinical trial. As a model tumor antigen, we selected the point-mutated (codon 12) Ki-Ras p21 oncogene (muRas) frequently (∼85%) present in pancreatic adenocarcinoma. Autologous LCLs were established in vitro by spontaneous outgrowth from peripheral blood lymphocytes of seven pancreatic carcinoma patients and were genetically modified with an episomal Epstein-Barr virus (EBV)-based expression vector to express muRas (muRas-LCL). Weekly vaccinations with subcutaneous injection of 5×10(6) muRas-LCL were done. In six of seven patients, therapeutic vaccination elicited a T-cell response with an increase in the frequency of muRas-specific precursor cytotoxic T lymphocytes in the peripheral blood and positive delayed-type hypersensitivity reactions at the injection site. Besides local reactions and flu-like symptoms, there were no signs of toxicity and no acute EBV infection, onset of EBV-associated lymphoma, or other severe complications. A clinical response (stable disease) was observed for a short time period (2-4 months) in four of seven patients (57%), mostly in earlier tumor stages. Our results indicate that LCL presenting genetically modified antigen represent a valuable and easily available tool for in vivo autologous tumor vaccination. LCL can be transfected with any known tumor antigen and therefore should be further clinically investigated.

A peptide epitope derived from the cancer testis antigen HOM-MEL-40/SSX2 capable of inducing CD4⁺ and CD8⁺ T-cell as well as B-cell responses.

BACKGROUND: Antigen-derived HLA class I-restricted peptides can generate specific CD8(+) T-cell responses in vivo and are therefore often used as vaccines for patients with cancer. However, only occasional objective clinical responses have been reported suggesting the necessity of CD4(+) T-cell help and possibly antibodies for the induction of an effective anti-tumor immunity in vivo. The SSX2 gene encodes the cancer testis antigen (CTA) HOM-MEL-40/SSX2, which is frequently expressed in a wide spectrum of cancers. Both humoral and cellular immune responses against SSX2 have been described making SSX2 an attractive candidate for vaccine trials. METHODS: SYFPEITHI algorithm was used to predict five pentadecamer peptides with a high binding probability for six selected HLA-DRB1 subtypes (*0101, *0301, *0401, *0701, *1101, *1501) which are prevalent in the Caucasian population. RESULTS: Using peripheral blood cells of 13 cancer patients and 5 healthy controls, the HOM-MEL-40/SSX2-derived peptide p101-111 was identified as an epitope with dual immunogenicity for both CD4(+) helper and cytotoxic CD8(+) T cells. This epitope also reacted with anti-SSX2 antibodies in the serum of a patient with breast cancer. Most remarkably, SSX2/p101-111 simultaneously induced specific CD8, CD4, and antibody responses in vitro. CONCLUSIONS: p101-111 is the first CTA-derived peptide which induces CD4(+), CD8(+), and B-cell responses in vitro. This triple-immunogenic peptide represents an attractive vaccine candidate for the induction of effective anti-tumor immunity.

Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad antibody responses in humans, a RAYS-based analysis.

Antibody responses to tumor antigens play an important role in initiating a cellular antitumor response with respect to antigen cross-presentation and T cell cross-priming. Successful vaccination strategies rely on an optimally timed activation of the humoral and cellular immune system by using appropriate adjuvant stimulation. The LUD99-008 trial used the cancer testis antigen NY-ESO-1 formulated with ISCOMATRIX adjuvant injected into patients intramuscularly. It was shown that this vaccination strategy is a safe and highly potent activator of the humoral and cellular immune system. Using the RAYS technology, we analyzed in detail the humoral immune response in these patients before and after vaccination: during the course of repeated vaccinations with the adjuvant, antibody titers against NY-ESO-1 and cross-reactivity to LAGE 1A and B increased as an indicator of an enhanced immune response, whereas no antibody response could be detected after vaccination without the adjuvant. Analysis of single fragments of the NY-ESO-1 protein revealed that the humoral response was almost exclusively directed against the N-terminal fragments and the number of fragments and their length being recognized by the NY-ESO-1-specific antibodies increased during the course of vaccination. The humoral immune response mainly consisted of antibodies of the IgG1 and IgG3 subclass. We rarely found IgG2 and IgG4 subclass antibodies. Our results support the implication that target-specific antibodies raised after vaccination contribute to the stimulation of an effective T cell response against the target antigen.

Cryptic epitopes induce high-titer humoral immune response in patients with cancer.

In search of novel markers for diagnosis, prognosis, and therapy of cancer, screening of rcDNA expression libraries with patient's sera has been established as a valuable tool for identification of cancer-specific Ags. Interestingly, besides the expected humoral responses to annotated proteins, patients with cancer were frequently found to have serum Abs that bind to peptides without homology to known proteins. So far, the nature of these unconventional epitopes and their possible significance in tumor immunology have never been thoroughly investigated. In our study, we specifically analyzed humoral immune response toward such peptides in patients with pancreatic or breast cancer using yeast-displayed cDNA expression libraries derived from tumor tissue. A detailed analysis of the identified peptides revealed that they originated from translation of sequences outside annotated open reading frames and may derive from the use of alternative start codons or from DNA indel mutations. In several cases, the corresponding mRNA templates have a known association with cancer. In a final analysis, we were able to detect one of these tumor Ags in cancer tissue arrays by a selected Fab-Ab. We conclude that cryptic epitopes may elicit specific humoral immune responses in patients with cancer and thus play a role in immunologic surveillance. Due to the high prevalence of immune responses against some of the peptides, they may also be valuable markers for cancer diagnosis, prognosis, or therapy monitoring.

Analysis of the antibody repertoire of patients with mantle cell lymphoma directed against mantle cell lymphoma-associated antigens.

Treatment results of mantle cell lymphomas (MCL) are not satisfactory and novel therapeutic approaches are warranted. Because "shared" tumor antigens like the group of cancer testis antigens are only rarely expressed in MCL, we applied serological analysis of antigens using recombinant expression cloning (SEREX) to a complementary DNA library derived from five cases of MCL using the sera of eight patients with MCL in order to define MCL-associated antigens that are immunogenic in these patients and might be used as vaccines for patients with MCL. Five antigens were detected by SEREX. Four of the five detected antigens (hypothetical protein FLK10233, recombining binding protein suppressor, a chromosomal sequence, and interleukin-1 receptor associated kinase) are also expressed by a wide spectrum of normal human cells, excluding their use as vaccines. In contrast, the expression of CD52, which was detected by antibodies in the serum of an MCL patient, is restricted to hematopoietic cells. Interestingly, anti-CD52 antibodies were detected in this patient before and >2 years after allogeneic transplantation, indicating that both the autologous as well as the allogeneic immune system recognized CD52. Since the anti-CD52 monoclonal antibody alemtuzumab has shown activity in MCL, a vaccine consisting of recombinant CD52 alone or combined with passive immunotherapy using alemtuzumab warrants furthers clinical and immunological evaluation in MCL.

Spontaneous high-titered IgG antibody responses against BCL-2 in patients with aggressive lymphomas.

PURPOSE: Molecules which are exclusively or preferentially expressed by malignant cells are potential targets for immunotherapeutic approaches to the treatment of cancer. METHODS: We therefore tested serum samples of 95 patients with aggressive B-cell lymphomas for antibodies against lymphoma-associated molecules using SEREX for antibodies against BCL-2, BCL-XL, MCL-1, survivin, BCL-6, CD20, LM02, PDE4B, cyclin-D2, actinin-alpha1, SCYA3, PKCbeta2, E2IG3, and HGAL. RESULTS: One patient had low-titered (1:100) antibodies against PKCbeta2. Antibodies against BCL-2 were detected in the sera of five patients (5.3%), with responses found more frequently in follicular lymphoma grade 3 (3/26 or 11.5%) than in diffuse large B-cell lymphomas (2/67 or 3%). Anti-BCL-2 antibodies were of the IgG class and titers ranged from 1:1,000 to 1:100,000 with highest titers before treatment and decreasing slowly during remission. CONCLUSION: The spontaneous immunogenicity of BCL-2 makes this molecule a prime candidate for vaccine approaches in BCL-2 positive B-cell lymphomas.