Additional booster doses in patients with chronic lymphocytic leukemia induce humoral and cellular immune responses to SARS-CoV-2 similar to natural infection regardless ongoing treatments: A study by ERIC, the European Research Initiative on CLL.

Profound immune dysregulation and impaired response to the SARS-CoV-2 vaccine put patients with chronic lymphocytic leukemia (CLL) at risk of severe COVID-19. We compared humoral memory and T-cell responses after booster dose vaccination or breakthrough infection. (Green) Quantitative determination of anti-Spike specific antibodies. Booster doses increased seroconversion rate and antibody titers in all patient categories, ultimately generating humoral responses similar to those observed in the postinfection cohort. In detail, humoral response with overscale median antibody titers arose in >80% of patients in watch and wait, off-therapy in remission, or under treatment with venetoclax single-agent. Anti-CD20 antibodies and active treatment with BTK inhibitors (BTKi) represent limiting factors of humoral response, still memory mounted in ~40% of cases following booster doses or infection. (Blue) Evaluation of SARS-CoV-2-specific T-cell responses. Number of T-cell functional activation markers documented in each patient. The vast majority of patients, including those seronegative, developed T-cell responses, qualitatively similar between treatment groups or between vaccination alone and infection cases. These data highlight the efficacy of booster doses in eliciting T-cell immunity independently of treatment status and support the use of additional vaccination boosters to stimulate humoral immunity in patients on active CLL-directed treatments.

The frequency of T regulatory cells modulates the survival of multiple myeloma patients: detailed characterisation of immune status in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an immunoproliferative disease characterised by the uncontrolled proliferation of plasma cells, which is accompanied by defects in the immune system. METHODS: This study aimed to characterise the frequency of T regulatory cells (Tregs), dendritic cells (DCs) as well as sub-populations of T cells bearing regulatory properties like CD4(+)GITR(+), CD4(+)CD62L(+), CD3(+)TCRγδ(+) along with the concentrations of IL-10, TGFß, IL-6 in 66 patients with MM. Subsequently, the influence of therapy on those components of immune system was assessed. RESULTS: The percentage of both myeloid and plasmacytoid DC was lower in MM compared with control group while Treg (CD4(+)CD25(high)FOXP3(+)) frequencies were significantly higher in MM patients compared with healthy control (6.16% vs 0.05%, respectively). Also, the percentages of CD4(+)GITR(+), CD4(+)CD62L(+) were increased compared with healthy volunteers. We found that patients with higher percentages of Treg live shorter (median overall survival 21 months vs not-reached, P=0.013). CONCLUSION: This study identifies several abnormalities of immune system in MM, which only partly could be normalised after successful therapy. The dysfunction of immune system such as decreased antigen presentation along with increased frequencies of suppressive cells and cytokines might facilitate progression of the disease and infectious complications limiting survival of MM patients.

Gastrointestinal bleeding as initial presentation of melanoma of unknown primary origin: report of a case and review of the literature.

The case of an 81-year-old patient, initially presenting with gastrointestinal (GI) bleeding, including melena and hematemesis is reported. Endoscopy revealed an ulcerated mass of the stomach corpus with immunohistochemistry stains consistent with metastatic melanoma. The thorough physical and paraclinical examination did not reveal any lesions or nodules as a primary site of the disease. The literature concerning this rare presentation of melanoma is also reviewed.

The significance of soluble HLA-G plasma levels as well as messenger HLA-G for B-cell chronic lymphocytic leukemia (B-CLL).

The immunosuppression accompanies B-cell chronic lymphocytic leukemia (B-CLL) but might be also responsible for disease progression by enabling CLL cells to escape from the immunosurveillance. Some particles involved in the regulation of an immune system might represent prognostic value for B-CLL. Recently we found no correlation between HLA-G on messenger and protein level, suggesting that HLA-G is released in soluble form. To confront this hypothesis we characterized soluble HLA-G (sHLA-G) by the prognostic factors in the first cohort of 34 CLL patients. No correlation was observed between sHLA-G levels in ZAP-70(+) and ZAP-70(-) CLL as well as in CD38(+) CLL and CD38(-) CLL patients. Next, we wondered whether gene expression of HLA-G, which represent the whole HLA-G pool in the cell, posses prognostic value for CLL. In the second cohort of 41 CLL patients we assessed messenger levels of HLA-G by the strongest prognostic factors in CLL including cytogenetics, IgVH mutational status, ZAP-70 as well as CD38. No changes of HLA-G expression levels were found in different CLL groups characterized by IgVH gene mutational status, ZAP-70 as well as CD38. We observed no differences in expression of HLA-G in various cytogenetic groups of CLL including del17p, del13q, del11q, +8q, +3q, del14q and del6q when compared to those with normal karyotype or with 12+. Both, mRNA expression of HLA-G and levels of its soluble form in plasma bring no additional prognostic value for B-CLL patients.

Expression of RHAMM/CD168 and other tumor-associated antigens in patients with B-cell chronic lymphocytic leukemia.

Antigen targeted immunotherapies might represent a novel treatment for B-cell chronic lymphocytic leukemia (B-CLL). We screened the mRNA expression of tumor-associated antigens (TAAs) from the literature (fibromodulin, survivin, OFA-iLRP, BAGE, G250, MAGE1, PRAME, proteinase, syntaxin, hTERT, WT-1) and TAAs defined previously by serological analysis of cDNA expression libraries from leukemic cells (PINCH, HSJ2, MAZ, MPP11, RHAMM/CD168, NY-Ren60). Peripheral blood mononuclear cells from 43 B-CLL patients and 20 healthy volunteers (HVs) were examined by conventional and quantitative RT-PCR. mRNA of RHAMM/CD168, fibromodulin, syntaxin and NY-Ren60 was expressed in 55-90%, and mRNA of HSJ2, MAZ and OFAiLRP was expressed in 90-100% of the patients. No expression of WT-1, hTERT, BAGE, G250, MAGE1 or survivin was observed. Low (2-20%) expression frequencies of MPP11, PINCH, PRAME and proteinase were detected. RHAMM/CD168, fibromodulin, PRAME and MPP11 showed expression in B-CLL patients, but not in HVs. Because of the exquisite tissue expression of RHAMM/CD168 and its high expression frequency in CLL patients, mixed lymphocyte peptide culture (MLPC), enzyme-linked immunosorbent spot (ELISPOT) and flow cytometry were performed for antigen specific T-cells. In MLPC, RHAMM specific responses by CD8+HLA-A2/R3tetramer+CCR7-CD45RAhigh effector T-cells were detected. RHAMM/CD168 might be a possible target for future immunotherapies in both ZAP-70(+) and ZAP-70(-) B-CLL patients.

Allogeneic dendritic cells pulsed with tumor lysates or apoptotic bodies as immunotherapy for patients with early-stage B-cell chronic lymphocytic leukemia.

Recently, immunotherapies with allogeneic dendritic cells (DCs) pulsed with tumor antigens to generate specific T-cell responses have been tested in clinical trials for patients with solid tumors. This is the first report on a clinical vaccination study with DCs for patients with B-cell chronic lymphocytic leukemia (B-CLL). The potential of allogeneic DCs pulsed ex vivo with tumor cell lysates or apoptotic bodies to stimulate antitumor immunity in patients with B-CLL in early stages was evaluated. Monocyte-derived DCs were obtained from unrelated healthy donors. Nine patients (clinical stage 0 and 1 according to Rai) were vaccinated five times with a mean number of 32 x 10(6) stimulated DCs administered intradermally once every 2-3 weeks. No signs of autoimmunity were detected, and only mild local skin reactions were noted. During the treatment period, we observed a decrease of peripheral blood leukocytes and CD19+/CD5+ leukemic cells. In one patient, a significant increase of specific cytotoxic T lymphocytes against RHAMM/CD168, a recently characterized leukemia-associated antigen, could be detected after DC vaccination. Taken together, the study demonstrated that DC vaccination in CLL patients is feasible and safe. Immunological and to some extent hematological responses could be noted, justifying further investigation on this immuno-therapeutical approach.

Gene expression profiling in MDS and AML: potential and future avenues.

Today, the classification systems for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) already incorporate cytogenetic and molecular genetic aberrations in an attempt to better reflect disease biology. However, in many MDS/AML patients no genetic aberrations have been identified yet, and even within some cytogenetically well-defined subclasses there is considerable clinical heterogeneity. Recent advances in genomics technologies such as gene expression profiling (GEP) provide powerful tools to further characterize myeloid malignancies at the molecular level, with the goal to refine the MDS/AML classification system, incorporating as yet unknown molecular genetic and epigenetic pathomechanisms, which are likely reflected by aberrant gene expression patterns. In this study, we provide a comprehensive review on how GEP has contributed to a refined molecular taxonomy of MDS and AML with regard to diagnosis, prediction of clinical outcome, discovery of novel subclasses and identification of novel therapeutic targets and novel drugs. As many challenges remain ahead, we discuss the pitfalls of this technology and its potential including future integrative studies with other genomics technologies, which will continue to improve our understanding of malignant transformation in myeloid malignancies and thereby contribute to individualized risk-adapted treatment strategies for MDS and AML patients.

Definition of a target for immunotherapy and results of the first Peptide vaccination study in chronic lymphocytic leukemia.

Results of bone marrow transplantation, as well as remission phenomena after viral infections, suggest that chronic lymphocytic leukemia (CLL) might be targeted effectively by T-cell-based immunotherapy. Antigen-targeted immunotherapies represent novel treatments for CLL patients. Earlier, we screened the mRNA expression of several tumor associated antigens (TAAs), observing the presence of RHAMM/CD168, fibromodulin, syntaxin, and NY-Ren60 in 55%-90% of CLL patients. RHAMM/CD168, fibromodulin, PRAME, and MPP11 were expressed in CLL patients but not in healthy volunteers. Quantitative reverse transcriptase polymerase chain reaction revealed higher RHAMM expression in high-risk CLL patients as well as in advanced stages of the disease. CLL cases with higher RHAMM expressions showed significantly shorter median treatment-free survivals. Among patients with mutated IgVH genes, an analysis of RHAMM expression enabled us to distinguish a subgroup of patients with a favorable prognosis. In lymph nodes, RHAMM staining correlated with a higher Ki-67 index and CD40L expression. Functionally, stimulation with CD40L enhanced RHAMM expression in CLL. Because of the exquisite tissue expression of RHAMM and its high expression frequency in CLL patients, we further characterized RHAMM-specific CD8+ T cells in these patients. CD8+ T cells primed with the RHAMM-derived epitope R3, which is restricted by human leukocyte antigen (HLA)A2, lysed RHAMM+ CLL cells. Therefore, we initiated a Phase I clinical trial of R3 peptide vaccination. Four patients exhibited reduced white blood cell counts during the vaccination process. In 5/6 patients, R3-specific CD8+ T cells were detected with the corresponding peptide/HLA-A2 tetrameric complex; these populations were verified functionally in 4/5 patients using ELISpot assays. In conclusion, RHAMM expression seems to be of prognostic value, and may reflect the proliferative capacity of CLL cells; it may therefore represent an interesting target for immunotherapy. Peptide vaccination in CLL patients was safe eliciting specific CD8+ T-cell responses against the tumor antigen RHAMM.

Peptide vaccination elicits leukemia-associated antigen-specific cytotoxic CD8+ T-cell responses in patients with chronic lymphocytic leukemia.

The receptor for hyaluronic acid-mediated motility (RHAMM) is a tumor-associated antigen in chronic lymphocytic leukemia (CLL). CD8(+) T cells primed with the RHAMM-derived epitope R3, which is restricted by human leukocyte antigen (HLA)-A2, effectively lyse RHAMM(+) CLL cells. Therefore, we initiated a phase I clinical trial of R3 peptide vaccination. Six HLA-A2(+) CLL patients were vaccinated four times at biweekly intervals with the R3 peptide (ILSLELMKL; 300 microg per dose) emulsified in incomplete Freund's adjuvant; granulocyte-macrophage colony stimulating factor (100 microg per dose) was administered concomitantly. Detailed immunological analyses were conducted throughout the course of peptide vaccination. No severe adverse events greater than CTC I degrees skin toxicity were observed. Four patients exhibited reduced white blood cell counts during vaccination. In five of six patients, R3-specific CD8(+) T cells were detected with the corresponding peptide/HLA-A2 tetrameric complex; these populations were verified functionally in four of five patients using enzyme-linked immunosorbent spot (ELISpot) assays. In patients with clinical responses, we found increased frequencies of R3-specific CD8(+) T cells that expressed high levels of CD107a and produced both interferon-gamma and granzyme B in response to antigen challenge. Interestingly, vaccination was also associated with the induction of regulatory T cells in four patients. Thus peptide vaccination in six CLL patients was safe and could elicit to some extent specific CD8(+) T-cell responses against the tumor antigen RHAMM.

Thalidomide exerts distinct molecular antileukemic effects and combined thalidomide/fludarabine therapy is clinically effective in high-risk chronic lymphocytic leukemia.

Thalidomide represents a promising immunomodulatory drug that targets both leukemia cells and the tumor microenvironment. We treated patients with chronic lymphocytic leukemia (CLL) with a combined thalidomide/fludarabine regimen and monitored cellular and molecular changes induced by thalidomide in vivo before fludarabine treatment. Thalidomide was given daily (100 mg p.o. per day) and fludarabine was administered on days 7-11 (25 mg/m(2) i.v. per day) within each 4-week cycle (maximum of 6 cycles). Twenty patients received thalidomide/fludarabine as first-line therapy and 20 patients were previously treated. Unmutated IgVH mutation status was found in 36 cases and 13 had high-risk cytogenetic aberrations (del17p, del11q). The overall response rate was 80 and 25% for untreated and previously treated patients, respectively. Although thalidomide reduced the number of CLL cells, the number of CD3 lymphocytes showed no significant change, but the number of CD4(+)CD25(hi)FOXP3(+) regulatory T cells (Tregs) was significantly decreased. Gene expression profiling revealed a thalidomide-induced signature containing both targets known to have a function in immunomodulatory drug action as well as novel candidate genes. Combined thalidomide/fludarabine therapy demonstrated efficacy in high-risk patients with CLL. Furthermore, our study provides novel biological insights into thalidomide effect, which might act by enhancing apoptosis of CLL cells and reducing Tregs, thereby enabling T-cell-dependent antitumor effect.

The candidate immunotherapeutical target, the receptor for hyaluronic acid-mediated motility, is associated with proliferation and shows prognostic value in B-cell chronic lymphocytic leukemia.

Differential expression of molecules in chronic lymphocytic leukemia (CLL) may define prognostic markers and suitable targets for immunotherapy. Expression of the tumor-associated antigen (TAA) RHAMM (receptor for hyaluronic acid-mediated motility) as well as RHAMM splicing variants was assessed in series of 72 CLL patients. Quantitative reverse transcriptase PCR showed higher RHAMM expression in high-risk CLL patients, as well as in the advanced stages of the disease. CLL cases with a higher RHAMM expression showed a significantly shorter median treatment-free survival. Among patients with mutated immunoglobulin heavy chain genes, an analysis of RHAMM expression enabled to distinguish subgroup of patients with favorable prognosis. In lymph nodes, RHAMM staining correlated with a higher Ki-67 index and CD40L expression. Functionally, stimulation with CD40L enhanced RHAMM expression in CLL. We further characterized RHAMM-specific CD8(+) T cells in patients with CLL, as the expression of TAAs might influence the clinical outcome by the means of immune reactions. The cytotoxic potential of RHAMM-specific T cells was shown against target cells bearing RHAMM-derived epitope as well as against CLL cells expressing RHAMM. In conclusion, RHAMM expression appears to be of prognostic value, as well as may reflect the proliferative capacity of CLL cells, and might therefore represent interesting target for immunotherapy.

Expression of HLA-G in patients with B-cell chronic lymphocytic leukemia (B-CLL).

The expression of HLA-G was reported in certain malignancies and its role in escaping from immunosurveillance in cancers was proposed since HLA-G is a nonconventional HLA class I molecule that protects fetus from immunorecognition during pregnancy. Recent studies proposed HLA-G as novel prognostic marker for patients with B-CLL. HLA-G was showed to bear even better prognostic information compared to Zeta-chain associated protein of 70kDa (ZAP-70) and CD38 although some other authors did not find HLA-G expression in CLL. Therefore in this study we characterized the expression of HLA-G on both RNA and protein level. In most of 20 B-CLL patients we were able to detect signal from HLA-G using flow cytometry analysis. The expression of HLA-G was confirmed on messenger level by real-time RT-PCR experiments. No correlation between HLA-G expression and expression of well established prognostic factors such as ZAP-70 and CD38 was detected. These results confirm that HLA-G is expressed on CLL leukemic cells. Furthermore the expression of HLA-G on CLL cells suggests that this molecule might be involved in escaping of CLL cells from immunosurveillance.

Vaccination of B-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response.

Recently, we described that vaccination with allogeneic dendritic cells (DCs) pulsed with tumor cell lysate generated specific CD8+ T cell response in patients with B-cell chronic lymphocytic leukemia (B-CLL). In the present study, the potential of autologous DCs pulsed ex vivo with tumor cell lysates to stimulate antitumor immunity in patients with B-CLL in early stages was evaluated. Twelve patients at clinical stage 0-2 as per Rai were vaccinated intradermally up to eight times with a mean number of 7.4 x 10(6) DCs pulsed with B-CLL cell lysate. We observed a decrease of peripheral blood leukocytes and CD19+/CD5+ leukemic cells in five patients, three patients showed a stable disease and four patients progressed despite DC vaccination. A significant increase of specific cytotoxic CD8+ T lymphocytes against the leukemia-associated antigens RHAMM or fibromodulin was detected in four patients after DC vaccination. In patients with a clinical response, an increase of interleukin 12 (IL-12) serum levels and a decrease of the frequency of CD4+CD25(+)FOXP3+ T regulatory cells were observed. Taken together, the study demonstrated that vaccination with autologous DC in CLL patients is feasible and safe. Immunological and to some extend hematological responses could be noted, justifying further investigation on this immunotherapeutical approach.

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays.

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8(+) T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

Monoclonal antibodies.

Monoclonal antibodies are essential tools for many molecular immunology investigations. In particular, when used in combination with techniques such as epitope mapping and molecular modelling, monoclonal antibodies enable the antigenic profiling and visualisation of macromolecular surfaces. In addition, monoclonal antibodies have become key components in a vast array of clinical laboratory diagnostic tests. Their wide application in detecting and identifying serum analytes, cell markers, and pathogenic agents has largely arisen through the exquisite specificity of these unique reagents. Furthermore, the continuous culture of hybridoma cells that produce these antibodies offers the potential of an unlimited supply of reagent. In essence, when compared with the rather limited supply of polyclonal antibody reagents, the feature of a continuous supply enables the standardisation of both the reagent and the assay technique. Clearly, polyclonal and monoclonal antibodies have their advantages and disadvantages in terms of generation, cost, and overall applications. Ultimately, monoclonal antibodies are only produced when necessary because their production is time consuming and frustrating, although greatly rewarding (at least most of the time!). This is especially apparent when a monoclonal antibody can be applied successfully in a routine pathology laboratory or can aid in the clinical diagnosis and treatment of patients. In this article, the generation and application of monoclonal antibodies are demystified to enable greater understanding and hopefully formulate novel ideas for clinicians and scientists alike.