Anne Letsch

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.

Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity

Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity

Chemokine Receptor CCR6 Expression Level and Liver Metastases in Colorectal Cancer

PURPOSE The liver is the primary organ of metastasis in colorectal cancer (CRC). Chemokine receptor CCR6 is expressed on a subset of T cells and is associated with their migration into the liver. This study was performed to analyze a possible association between CCR6 expressed by primary CRC and liver metastases. PATIENTS AND METHODS CCR6 expression levels were evaluated by immunohistology in 64 CRC primary tumor specimens. Twenty-four of 64 patients had synchronous liver metastases. Evaluation of immunostaining was performed semiquantitatively by visual assessment and quantitatively by digital image analysis (DIA). Multiple logistic regression analysis was performed to assess relevant parameters for liver metastases. RESULTS CCR6 expression was verified in all 64 primary tumor specimens with considerable variations in intensity; 21 tumors (33%) demonstrated weak CCR6 staining, 32 (50%) demonstrated intermediate staining, and 11 (17%) demonstrated strong staining. Quantitative assessment by DIA showed an up to 5-log difference in CCR6 values. CCR6 staining was significantly stronger in tumor cells compared with adjacent colon epithelial cells (P < .0005). Multiple logistic regression analysis, controlling for age, sex, tumor stage, nodal status, pathologic grade, and preoperative carcinoembryonic antigen levels, revealed that CCR6 staining in the primary tumor was independently associated with the presence of liver metastases (odds ratio = 2.1; P = .002). CONCLUSION The association between expression level of CCR6 in primary CRC and synchronous liver metastases suggests that CCR6 and its ligand may be involved in the metastatic spread to the liver. Therefore, CCR6 may be a potential target for specific therapeutic interventions.

Quantitative Detection of Circulating Tumor Cells in Cutaneous and Ocular Melanoma and Quality Assessment by Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Purpose: Inconsistent reports on the detection of melanoma cells in peripheral blood by reverse transcriptase-PCR (RT-PCR) have resulted in uncertainty on the prognostic value of circulating melanoma cells. Experimental Design: We developed real-time RT-PCR assays for quantitation of tyrosinase, MelanA/MART1, and gp100 and for porphobilinogen deaminase housekeeping gene. Melanoma tissue (n = 18), peripheral blood samples from healthy donors (n = 21), and patients with cutaneous (n = 122) and uveal (n = 64) melanoma from our institution were analyzed. For quality control, an additional 251 samples from ongoing multicenter studies were compared with in-house samples. Results: Tyrosinase was not detected in healthy donor blood samples. For the two other markers, cutoff values had to be defined to distinct patient samples from controls. Patients with stage IV uveal and cutaneous melanoma expressed all three markers more frequently and at higher levels in peripheral blood as compared with earlier stages. The variation of expression was 4 logs and correlated with tumor load and serum lactate dehydrogenase. In 2 of 3 uveal melanoma patients, detection of circulating tumor cells preceded the development of liver metastases. The diagnostic sensitivity was optimal in blood samples containing >0.1pg/μl porphobilinogen deaminase (95.7% of in-house samples and 57.4% of multicenter samples). Conclusions: Real-time RT-PCR is able to quantitatively define the quality of a sample and provides quantitative data for melanoma markers. Disparities in the results of previous studies may be attributable to undetected differences in sample quality. The prognostic relevance of this assay is currently under evaluation in several prospective randomized trials.

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.

CMV‐specific central memory T cells reside in bone marrow

CMV‐specific CD8+ T cell responses in peripheral blood (PB) are characterized by a preponderance of effector and effector memory T cells. CMV‐specific central memory T cells (TCM), which are considered crucial in maintaining long‐term immunity, are rarely detectable in PB. In this study we have analyzed differentiation and function of CMV pp65‐specific CD8+ T cells in paired samples of human PB and BM using intracellular cytokine and tetramer staining. Overall frequencies of CMV pp65‐specific T cells were similar in PB compared to BM; however, CMV‐specific CD45RA–CCR7+ TCM were almost exclusively detectable in BM, which was not related to a general accumulation of TCM in BM. In vitro, CMV‐specific T cells could be more efficiently expanded from BM (median 128‐fold, n=6) than from PB (median 72‐fold, p=0.01). Taken together, these data show that the BM is a compartment harboring CMV‐specific TCM and underline the concept of the BM as a secondary immune organ. CMV specific BM‐derived TCM might be a valuable source for generating T cells for adoptive transfer.

Differences in T-cell immunity toward tumor-associated antigens in colorectal cancer and breast cancer patients.

There is increasing evidence that tumors elicit specific T‐cell responses in a substantial proportion of patients. Recently, we have shown that in patients with colorectal cancer specific T cells against the tumor‐associated antigens (TAA) Ep‐CAM, her‐2/neu or CEA can be detected in peripheral blood using IFNγ‐ELISPOT assay. In our study, we have analyzed T‐cell responses against HLA‐A*0201‐restricted epitopes of these TAA in peripheral blood of patients with breast cancer and colorectal cancer. Surprisingly, a complete absence of ex vivo T‐cell responses against these TAA was found in 20 patients with breast cancer. In contrast, specific T cells were detectable in 12 of 49 patients with colorectal cancer against at least 1 of these TAA, confirming our previous results. T‐cell responses against influenza‐derived peptides were similar in both malignancies. The results of our study indicate a difference either of tumor immunogenicity or of the migratory pattern of tumor‐specific T cells between breast cancer and colorectal cancer patients. The findings reported here have implications for the development of antigen‐specific T‐cell therapies.

T cell responses against tumor associated antigens and prognosis in colorectal cancer patients.

IntroductionSpontaneous T cell responses against specific tumor-associated antigens (TAA) are frequently detected in peripheral blood of tumor patients of various histiotypes. However, little is known about whether these circulating, spontaneously occurring, TAA-reactive T cells influence the clinical course of disease.MethodsFifty-four HLA-A2 positive colorectal cancer patients had been analyzed for the presence of T cell responses against epitopes derived from the TAA Ep-CAM, her-2/neu, and CEA either by ELISPOT assay or by intracellular cytokine staining. Then, Kaplan-Meier survival analysis was performed comparing T-cell-responders and T-cell-non-responders. For comparison, a group of T-cell-non-responders was compiled stringently matched to T-cell-responders based on clinical criteria and also analyzed for survival.ResultsSixteen out of 54 patients had a detectable T cell response against at least one of the three tested TAA. Two out of 21 patients (9.5%) with limited stage of disease (UICC I and II) and 14 out of 33 patients (42.4%) with advanced disease (UICC III and IV) were T cell response positive. Comparing all T-cell-responders (n = 16) and all T-cell-non-responders (n = 38), no survival difference was found. In an attempt to reduce the influence of confounding clinical factors, we then compared 16 responders and 16 non-responders in a matched group survival analysis; and again no survival difference was found (p = 0.7).ConclusionIn summary, we found no evidence that spontaneous peripheral T cell responses against HLA-A2-binding epitopes of CEA, her-2/neu and Ep-CAM are a strong prognostic factor for survival.

Wilms Tumor Protein 1 (WT1) peptide vaccination-induced complete remission in a patient with acute myeloid leukemia is accompanied by the emergence of a predominant T-cell clone both in blood and bone marrow.

Within the last few years, the first peptide vaccination trials for treatment of acute myeloid leukemia (AML) have been initiated. Athough the presence of epitope-specific T cells could be seen both in bone marrow (BM) and peripheral blood (PB), nothing is known about their clonal composition. In this study, we analyzed material from a patient with recurrent AML vaccinated with “Wilms Tumor Protein 1” (WT1) peptide, who achieved a complete remission (CR) lasting for 12 months. For identification of expanded WT1-specific T-cell clones, enrichment by tetramer and IFN&ggr; secretion were followed by comparative quantitative reverse transcribed PCR (qRT PCR) quantification of all TCR V&bgr;-families. V&bgr;-families with increase in the enriched fraction were cloned and sequenced. A predominant clone was quantified by clonotypic qRT PCR from PB and BM. Quantity and functionality of WT1-specific cells were assessed by tetramer analyses and intracellular IFN&ggr; staining. A specific predominant clone was identified during clinical remission. Clone-specific qRT PCR showed an increase both in PB and BM after 8 vaccinations. Six months after achieving CR, the transcript levels in BM decreased. Relapse was accompanied by secondary rise of the WT1-specific clone in PB but not in BM. In parallel, a lack of vaccine-induced WT1 specific IFN&ggr; production was observed at that timepoint. In conclusion, we provide first data regarding evolution and compartmentalization of a peptide vaccine-induced T-cell clone in PB and BM of an AML patient. At the time of relapse, the same clone reappeared spontaneously in PB but not in BM showing impaired functionality.

Human peripheral blood and bone marrow Epstein–Barr virus-specific T-cell repertoire in latent infection reveals distinct memory T-cell subsets

EBV infection leads to life‐long viral persistence. Although EBV infection can result in chronic disease and malignant transformation, most carriers remain disease‐free as a result of effective control by T cells. EBV‐specific IFN‐γ‐producing T cells could be demonstrated in acute and chronic infection as well as during latency. Recent studies, however, provide evidence that assessing IFN‐γ alone is insufficient to assess the quantity and quality of a T‐cell response. Using overlapping peptide pools of latent EBV nuclear antigen 1 and lytic BZLF‐1 protein and multicolor flow cytometry, we demonstrate that the majority of ex vivo EBV‐reactive T cells in healthy virus carriers are indeed IL‐2‐ and/or TNF‐producing memory cells, the latter being significantly more frequent in BM. After in vitro expansion, a substantial number of EBV‐specific CD4+ and CD8+ T cells retained a CC‐chemokine receptor 7 (CCR7)‐positive memory phenotype. Based on their cytokine profiles, six different EBV‐specific T‐cell subsets could be distinguished with TNF‐single or TNF/IL‐2‐double producing cells expressing the highest CCR7 levels resembling early‐differentiated memory T cells. Our study delineates the memory T‐cell profile of a protective immune response and provides a basis for analyzing T‐cell responses in EBV‐associated diseases.