Deep sequencing of bone marrow microenvironments of patients with del(5q) myelodysplastic syndrome reveals imprints of antigenic selection as well as generation of novel T-cell clusters as a response pattern to lenalidomide.

In myelodysplastic syndromes with a partial deletion of the long arm of chromosome 5, del(5q), lenalidomide is believed to reverse anergic T-cell immunity in the bone marrow resulting in suppression of the del(5q) clone. In this study we used next-generation sequencing of immunoglobulin heavy chain (IGH) and T-cell receptor beta (TRB) rearrangements in bone marrow-residing and peripheral blood-circulating lymphocytes of patients with del(5q) myelodysplastic syndromes to assess the immune architecture and track adaptive immune responses during treatment with lenalidomide. The baseline bone marrow B-cell space in patients was comparable to that of age-matched healthy controls in terms of gene usage and IGH clonality, but showed a higher percentage of hypermutated IGH sequences, indicating an expanded number of antigen-experienced B lineage cells. Bone marrow B lineage clonality decreased significantly and hypermutated IGH clones normalized upon lenalidomide treatment, well in line with the proliferative effect on healthy antigen-inexperienced B-cell precursors previously described for this drug. The T-cell space in bone marrow of patients with del(5q) myelodysplastic syndromes showed higher TRB clonality compared to that of healthy controls. Upon lenalidomide treatment, myelodysplastic syndrome-specific T-cell clusters with low to medium spontaneous generation probabilities emerged; these clusters were shared across patients, indicating a common antigen-driven T-cell response pattern. Hence, we observed B lineage diversification and generation of new, antigen-dependent T-cell clusters, compatible with a model of adaptive immunity induced against the del(5q) clone by lenalidomide. Overall, this supports the concept that lenalidomide not only alters the functional T-cell state, but also the composition of the T- and B-cell repertoires in del(5q) myelodysplastic syndromes.

T-cell diversification reflects antigen selection in the blood of patients on immune checkpoint inhibition and may be exploited as liquid biopsy biomarker.

Cancer immunotherapy with antibodies targeting immune checkpoints, such as programmed cell death protein 1 (PD-1), shows encouraging results, but reliable biomarkers predicting response to this costly and potentially toxic treatment approach are still lacking. To explore an immune signature predictive for response, we performed liquid biopsy immunoprofiling in 18 cancer patients undergoing PD-1 inhibition before and shortly after initiation of treatment by multicolor flow cytometry and next-generation T- and B-cell immunosequencing (TCRß/IGH). Findings were correlated with clinical outcomes. We found almost complete saturation of surface PD-1 on all T-cell subsets after the first dose of the antibody. Both T- and B-cell compartments quantitatively expanded during treatment. These expansions were mainly driven by an increase in the activated T-cell compartments, as well as of naïve B- and plasma cells. Deep immunosequencing revealed a clear diversification pattern of the clonal T-cell space indicative of antigenic selection in 47% of patients, while the remaining patients showed stable repertoires. 43% of the patients with a diversification pattern showed disease control in response to the PD-1 inhibitor. No disease stabilizations were observed without clonal T-cell space diversification. Our data show for the first time a clear impact of PD-1 targeting not only on circulating T-cells, but also on B-lineage cells, shedding light on the complexity of the anti-tumor immune response. Liquid biopsy T-cell next-generation immunosequencing should be prospectively evaluated as part of a composite response prediction biomarker panel in the context of clinical studies.

A Combined Cyto- and Histopathological Diagnostic Approach Reduces Time to Diagnosis and Time to Therapy in First Manifestation of Metastatic Spinal Disease: A Cohort Study.

Malignant spinal lesions (MSLs) are frequently the first manifestation of malignant disease. Spinal care, diagnostic evaluation, and the initiation of systemic therapy are crucial for outcomes in patients (pts) with advanced cancer. However, histopathology (HP) may be time consuming. The additional evaluation of spinal lesions using cytopathology (CP) has the potential to reduce the time to diagnosis (TTD) and time to therapy (TTT). CP and HP specimens from spinal lesions were evaluated in parallel in 61 pts (CP/HP group). Furthermore, 139 pts in whom only HP was performed were analyzed (HP group). We analyzed the TTD of CP and HP within the CP/HP group. Furthermore, we compared the TTD and TTT between the groups. The mean TTD in CP was 1.7 ± 1.7 days (d) and 8.4 ± 3.6 d in HP (p < 0.001). In 13 pts in the CP/HP group (24.1%), specific therapy was initiated based on the CP findings in combination with imaging and biomarker results before completion of HP. The mean TTT in the CP/HP group was 21.0 ± 15.8 d and was significantly shorter compared to the HP group (28.6 ± 23.3 d) (p = 0.034). Concurrent CP for MSLs significantly reduces the TTD and TTT. As a result, incorporating concurrent CP for analyzing spinal lesions suspected of malignancy might have the potential to enhance pts' quality of life and prognosis in advanced cancer. Therefore, we recommend implementing CP as a standard procedure for the evaluation of MSLs.

Surgical Site Cytology to Diagnose Spinal Lesions.

Patients with new-onset malignant spinal lesions often have an urgent need for local spine intervention and systemic therapy. For optimal management, it is crucial to diagnose the underlying disease as quickly and reliably as possible. The aim of our current study was to determine the feasibility, sensitivity, specificity, and diagnostic certainty of complementary cytological evaluation of spinal lesions suspected of malignancy. In 44 patients, we performed histopathological biopsies and in parallel cytologic preparations from the malignant site. Cytological smears were prepared and stained for May-Grunwald and Giemsa. Bone biopsies were histopathologically analyzed according to the existing standard-of-care practices. In 42 of 44 cases (95%), a cytological sample was successfully obtained. In 40 cases (95.2%, Cohen's kappa: 0.77), the cytological diagnosis agreed with the histological diagnosis regarding the identification of a malignant lesion. This resulted in a sensitivity of 97% and a specificity of 80% as well as a diagnostic safety of 95%. Cytological analysis in the context of spinal surgery proved sufficient to establish a diagnosis of malignancy or its exclusion, expanding the existing diagnostic spectrum. Furthermore, implementation of this process as a routine clinical diagnostic might shorten the time to diagnosis and improve the treatment of this vulnerable patient group.

Addition of isatuximab to lenalidomide, bortezomib, and dexamethasone as induction therapy for newly diagnosed, transplantation-eligible patients with multiple myeloma (GMMG-HD7): part 1 of an open-label, multicentre, randomised, active-controlled, phase 3 trial.

BACKGROUND: Anti-CD38 monoclonal antibodies have consistently shown increased efficacy when added to standard of care for patients with multiple myeloma. We aimed to assess the efficacy of isatuximab in addition to lenalidomide, bortezomib, and dexamethasone in patients with newly diagnosed transplantation-eligible multiple myeloma. METHODS: This open-label, multicentre, randomised, active-controlled, phase 3 trial was done at 67 academic and oncology practice centres in Germany. This study is ongoing and divided into two parts; herein, we report results from part 1. Eligible patients were aged 18-70 years; had a confirmed diagnosis of untreated multiple myeloma requiring systemic treatment and a WHO performance status of 0-2; and were eligible for induction therapy, high-dose melphalan and autologous haematopoietic stem-cell transplantation, and maintenance treatment. Patients were randomly assigned (1:1) to receive three 42-day cycles of induction therapy either with isatuximab plus lenalidomide, bortezomib, and dexamethasone (isatuximab group) or lenalidomide, bortezomib, and dexamethasone alone (control group) using a web-based system and permuted blocks. Patients in both groups received lenalidomide (25 mg orally on days 1-14 and 22-35), bortezomib (1·3 mg/m2 subcutaneously on days 1, 4, 8, 11, 22, 25, 29, and 32), and dexamethasone (20 mg orally on days 1-2, 4-5, 8-9, 11-12, 15, 22-23, 25-26, 29-30, and 32-33). Isatuximab was given as 10 mg/kg intravenously on days 1, 8, 15, 22, and 29 of cycle 1 and on days 1, 15, and 29 of cycles 2 and 3. The primary endpoint was minimal residual disease (MRD) negativity assessed by flow cytometry, in the intention-to-treat (ITT) population. This study is registered with ClinicalTrials.gov, NCT03617731. FINDINGS: Between Oct 23, 2018, and Sep 22, 2020, 660 patients were included in the ITT analysis (331 in the isatuximab group and 329 in the control group). 654 (99%) patients were White, two were African, one was Arabic, and three were Asian. 250 (38%) were women and 410 (62%) were men. The median age was 59 years (IQR 54-64). MRD negativity after induction therapy was reached in 166 (50%) patients in the isatuximab group versus 117 (36%) in the control group (OR 1·82 [95% CI 1·33-2·48]; p=0·00017). Median follow-up time from start to end of induction therapy was 125 days (IQR 125-131) versus 125 days (125-132). At least one grade 3 or 4 adverse event occurred in 208 (63%) of 330 patients versus 199 (61%) of 328 patients. Neutropenia of grade 3 or 4 occurred in 77 (23%) versus 23 (7%) patients and infections of grade 3 or 4 occurred in 40 (12%) versus 32 (10%) patients. Among 12 deaths during induction therapy, one death due to septic shock in the isatuximab group and four deaths (one cardiac decompensation, one hepatic and renal failure, one cardiac arrest, and one drug-induced enteritis) in the control group were considered treatment-related. INTERPRETATION: Addition of isatuximab to lenalidomide, bortezomib, and dexamethasone for induction therapy improved rates of MRD negativity with no new safety signals in patients with newly diagnosed transplantation-eligible multiple myeloma. FUNDING: Sanofi and Bristol Myers Squibb (Celgene).

A clinical and immunologic phase 2 trial of Wilms tumor gene product 1 (WT1) peptide vaccination in patients with AML and MDS.

This study investigated the immunogenicity of Wilms tumor gene product 1 (WT1)-peptide vaccination in WT1-expressing acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients without curative treatment option. Vaccination consisted of granulocyte-macrophage colony-stimulating factor subcutaneously days 1 to 4, and WT1.126-134 peptide and 1 mg keyhole limpet hemocyanin on day 3. The initial 9 patients received 4 vaccinations biweekly, then monthly, and the subsequent 10 patients received continual biweekly vaccination. Seventeen AML patients and 2 refractory anemia with excess blasts patients received a median of 11 vaccinations. Treatment was well tolerated. Objective responses in AML patients were 10 stable diseases (SDs) including 4 SDs with more than 50% blast reduction and 2 with hematologic improvement. An additional 4 patients had clinical benefit after initial progression, including 1 complete remission and 3 SDs. WT1 mRNA levels decreased at least 3-fold from baseline in 35% of patients. In 8 of 18 patients, WT1-tetramer(+) T cells increased in blood and in 8 of 17 patients in bone marrow, with a median frequency in bone marrow of 0.18% at baseline and 0.41% in week 18. This WT1 vaccination study provides immunologic, molecular, and preliminary evidence of potential clinical efficacy in AML patients, warranting further investigations.

Vaccination strategies in patients with renal cell carcinoma.

Although new treatment options for patients with advanced renal cell cancer (RCC) have been developed within recent years, vaccination is still a promising emerging treatment option. An increasing number of tumor-associated antigens (TAA) available for RCC are currently used and analyzed for their efficacy for antigen-specific vaccine strategies. Recently, antigen-specific vaccination with dendritic cells in patients with metastatic RCC was shown to induce cytotoxic T cell response associated with objective clinical responses in some of the patients. Furthermore, current studies focus on the development of more effective vaccine regimes, such as the application of polyvalent, HLA-independent RNA coding for multiple TAA and adjuvants. First results demonstrate promising clinical and immunological efficacy. The efficacy of antigen-specific vaccination might be improved by a combination of tyrosine kinase inhibitors, since sunitinib was shown to promote T cell induction following vaccination in a mouse model and elimination of regulatory T cells.

Sunitinib treatment for patients with advanced clear-cell renal-cell carcinoma after progression on sorafenib.

BACKGROUND: Sorafenib and sunitinib are tyrosine kinase inhibitors with largely overlapping specificities, approved for the treatment of metastatic renal-cell carcinoma (RCC). It was unclear whether the similarities of the two drugs would lead to complete cross-resistance, or whether sequential application would be efficacious. METHODS: Patients with metastatic RCC and progression on sorafenib treatment were treated with repeated cycles of sunitinib, 50 mg for 4 weeks, followed by a 2-week break. Response (Response Evaluation Criteria in Solid Tumors, RECIST) was assessed every second cycle. RESULTS: A total of 22 patients with progression on sorafenib were accrued. Initially, sorafenib treatment was efficacious in all patients, with 7 showing partial response (PR) and 15 stable disease (SD), and subsequent disease progression. With 4 PRs (18%) and 12 SD (55%) a disease control rate of 73% was achieved. The median progression-free survival (PFS) on sunitinib was 21.5 weeks; median overall survival (OS) was not reached. Estimated 1-year PFS and OS were 31 and 60%, respectively. There was no apparent relationship between response to sorafenib and outcome on sunitinib. CONCLUSION: In our cohort of patients with RCC and progression after initial efficacy of sorafenib, the efficacy data of second-line sunitinib were close to published results of first-line treatment, suggesting limited clinically relevant cross-resistance.

Fulminant blast crisis with de novo 11q23 rearrangement in a Philadelphia-positive CML patient undergoing treatment with dasatinib.

BACKGROUND: Progression of chronic myeloid leukemia (CML) is frequently accompanied by cytogenetic evolution, with an extra copy of the Philadelphia chromosome, trisomy 8 and 19, and isochromosome (17p) commonly detected. Translocations involving 11q23 chromosomal region have been rarely reported in CML. The few reported patients with blast crisis (BC) of CML carrying an 11q rearrangement have insufficient responses to tyrosine kinase inhibitors (TKIs) and possess a poor prognosis. CASE REPORT: We report the case of a 30-year-old man with CML who had a fulminant myeloid BC 4 months after initiation of first-line therapy with the TKI dasatinib, despite showing an optimal response at the 3-month timepoint. Despite cytoreductive therapy with hydroxyurea and 3rd-generation TKI ponatinib, the patient died within 10 days after the diagnosis of BC. Cytogenetic analyses revealed additional genetic aberrations including trisomy 8 and t(9;11)(p21;q23) involving the mixed lineage leukemia (MLL) gene. CONCLUSION: The presence of 11q23 rearrangements in the relapse clone in BC of CML most likely accounts for the adverse clinical outcome. Thus, in the case of rapid and unexpected BC, the presence of 11q rearrangements should be tested together with other additional chromosomal alterations, and immediate addition of chemotherapy to the TKIs should be evaluated.

Identification of a highly immunogenic HLA-A*01-binding T cell epitope of WT1.

PURPOSE: The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of WT1 which could be useful for immunotherapy. EXPERIMENTAL DESIGN: Potential HLA-A*01 epitopes predicted by a MHC binding algorithm were screened for recognition by peripheral blood mononuclear cells (PBMC) from patients with spontaneous T cell responses using intracellular cytokine cytometry. Epitope processing was shown by proteasomal cleavage. Epitope-specific T cells were generated from CD4+CD25+ regulatory T cell-depleted PBMC. RESULTS: One of five predicted HLA-A*01-binding candidate epitopes showed high immunogenicity as 5 of 14 patients with hematologic malignancies had WT1.317-327-reactive T cells ranging from 0.4% to 1.5% of CD3+CD8+ T cells. Proteasomal degradation assays indicated the cleavage of WT1.317-327. The depletion of regulatory T cells from PBMCs enabled the rapid expansion of WT1.317-327-specific CTL, whereas no CTL could be generated from unfractionated PBMC. WT1.317-327-specific CTL efficiently lysed an autologous WT1-expressing tumor cell line but not HLA-A*01-negative WT1-expressing tumor cells. Immunogenicity of the epitope across histologies was verified by the demonstration of spontaneous ex vivo WT1.317-327-specific T cell responses in two of six patients with HLA-A*01-positive melanoma or lung cancer. CONCLUSION: In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*01-restricted T cell epitope of the tumor antigen WT1, which is of considerable interest for use in vaccination trials.

Addition of histamine to interleukin 2 treatment augments type 1 T-cell responses in patients with melanoma in vivo: immunologic results from a randomized clinical trial of interleukin 2 with or without histamine (MP 104).

PURPOSE: Preclinical investigations suggest that histamine dihydrochloride (HDC) protects T cells and natural killer cells from inhibition by monocyte-derived reactive oxygen metabolites and synergizes with interleukin (IL) 2 in inducing T-cell activation. Here, we investigate whether this mechanism is operational in patients with melanoma treated with HDC as an adjunct to IL-2. EXPERIMENTAL DESIGN: Melanoma patients having liver metastases were treated with IL-2 with or without HDC within a randomized, multicenter, phase III trial. The effect of HDC on type 1 and type 2 T-cell cytokine production was investigated in peripheral blood samples from 19 patients with the use of intracellular cytokine flow cytometry. Melanoma-specific T-cell responses were analyzed in eight HLA-A2-positive patients. RESULTS: Frequencies of CD3+ T cells producing IFN-gamma (type 1 T cells) in response to phorbol myristate acetate/ionomycin increased (median, 1.8-fold) in patients receiving IL-2 plus HDC but not in those receiving IL-2 alone (P < 0.01 for comparison between arms). In contrast, the number of IL-13-producing type 2 T cells that increased in patients after treatment with IL-2 was not modulated by HDC. Melanoma- and tyrosinase-specific IFN-gamma and IL-13-producing T cells were detected in two of four HLA-A2-positive patients with melanoma following treatment with HDC + IL-2. CONCLUSIONS: Treatment of patients with stage IV melanoma with HDC in combination with IL-2 increases type 1 T-cell responses and may promote induction of melanoma-specific T cells. These effects are of relevance for tumor immunotherapy and provide a potential mechanism for the clinical efficacy of HDC added to IL-2.

Circulating Tumor-reactive CD8(+) T cells in melanoma patients contain a CD45RA(+)CCR7(-) effector subset exerting ex vivo tumor-specific cytolytic activity.

To defend the host from malignancies, the immune system can spontaneously raise CD8(+) T-cell responses against tumor antigens. Investigating the functional state of tumor-reactive cytolytic T cells in cancer patients is a key step for understanding the role of these cells in tumor immunosurveillance and for evaluating the potential of immunotherapeutic approaches of vaccination against cancer. In this study we identified a subset of circulating tumor-reactive CD8(+) T lymphocytes, which specifically secreted IFN-gamma after exposition to autologous tumor cell lines in stage IV metastatic melanoma patients. Additional phenotypic characterization using multicolor flow cytometry revealed that a significant fraction of these cells were CD45RA(+)CCR7(-), a phenotype that has been proposed recently to characterize cytolytic effectors potentially able to home into inflamed tissues. In the case of an HLA-A2-expressing patient, the antigen specificity of this population was identified by using HLA-A2/peptide multimers incorporating a tyrosinase-derived peptide. Consistently with their phenotypic characteristics, A2/tyrosinase peptide multimer(+) CD8(+) T cells, isolated by cell sorting, were directly lytic ex vivo and able to specifically recognize tyrosinase-expressing tumor cells. Overall, these results provide the first evidence that a proportion of melanoma patients have circulating tumor-reactive T cells, which are lytic effectors cells.

Functional CCR9 expression is associated with small intestinal metastasis.

In general, metastases to the small intestine are rare, and mostly occur in melanoma. CCR9 has been shown to be the principal chemokine receptor for the thymus expressed chemokine (TECK), a chemokine selectively expressed in the small intestine and thymus. Here we show that CCR9 is highly expressed on melanoma cells and all melanoma cell lines isolated from small intestinal metastases, and on a proportion of cell lines from other sites. Only melanoma cells and cell lines from small intestinal metastases, however, were responsive to the CCR9 ligand TECK, as assessed by receptor downregulation and by actin polymerization. CCR9 expression was also found on the adenocarcinoma cell line CaCo-2 expressing characteristics of enterocytic differentiation, but not on any other cell line isolated from colorectal, breast, and lung cancer. Our data provide evidence that the aberrant functional cell surface expression of an organ-specific chemokine receptor is associated with metastasis to this site. The regulation of receptor function seems to be a critical step in the metastatic process.

Immunomodulatory effects of sorafenib on peripheral immune effector cells in metastatic renal cell carcinoma.

BACKGROUND: Tyrosine kinase inhibitors (TKI) such as sorafenib have substantially improved the prognosis of metastatic renal cell carcinoma (mRCC) patients, but long-term remissions have only been reached with immunotherapy. Sequencing or combining TKI treatment with immunotherapy may represent an attractive therapeutic concept. However, in vitro data have shown that TKI may not only affect tumour cells, but also inhibit signalling in immune effector cells. Therefore, we asked whether sorafenib had an influence on peripheral immune effector cells in a cohort of 35 mRCC patients receiving sorafenib treatment. METHODS: Peripheral blood (pB) samples were analysed at baseline and after 8 weeks of treatment. IL-10 and TGF-ß mRNA levels were quantified by RT-PCR; regulatory T cell (Treg) counts and intracellular cytokine responses (TNF-α, IFN-γ, IL-10 and TGF-ß) of mononuclear cell subsets were determined by flow cytometry after in vitro stimulation with PMA/ionomycin. RESULTS: Sorafenib did not alter the elevated TGF-ß and IL-10 mRNA levels or elevated frequencies of IL-10 and TGF-ß producing monocytes and had no influence on type 1 cytokine responses in pB. CD4+CD25(high) FOXP3+/CD3+ T cells, likely representing Treg cells, decreased during sorafenib therapy. CONCLUSIONS: In vivo, sorafenib treatment was associated with a decrease in frequency of Treg cells without influencing the function of peripheral immune effector cells. Therefore, although sorafenib did not convert the immunosuppressive phenotype associated with mRCC, it seemed to be a possible candidate for combination with immunotherapy.