ALBERT

Description: Although graft-versus-host disease (GVHD) is a life-threatening complication of hematopoietic stem-cell transplantation (HSCT), its current diagnosis depends mainly on clinical manifestations and invasive biopsies. Specific biomarkers for GVHD would facilitate early and accurate recognition of this grave condition. Using proteomics, we screened for plasma proteins specific for GVHD in a mouse model. One peak with 8972-Da molecular mass (m/z) retained a discriminatory value in 2 diagnostic groups (GVHD and normal controls) with increased expression in the disease and decreased expression during cyclosporin A treatment, and was barely detectable in syngeneic transplantation. Purification and mass analysis identified this molecule as CCL8, a member of a large chemokine family. In human samples, the serum concentration of CCL8 correlated closely with GVHD severity. All non-GVHD samples contained less than 48 pg/mL (mean ± SE: 22.5 ± 5.5 pg/mL, range: 12.6-48.0 pg/mL, n = 7). In sharp contrast, CCL8 was highly up-regulated in GVHD sera ranging from 52.0 to 333.6 pg/mL (mean ± SE: 165.0 ± 39.8 pg/mL, n = 7). Strikingly, 2 patients with severe fatal GVHD had extremely high levels of CCL8 (333.6 and 290.4 pg/mL. CCL8 is a promising specific serum marker for the early and accurate diagnosis of GVHD.

Description: Many studies have shown the presence of minimal residual disease (MRD) following therapy for childhood acute lymphoblastic leukemia (ALL) to be an important prognostic marker. We have also shown a significant relationship between survival outcomes in patients enrolled in the previous ALL 911 study and molecular MRD levels 5 weeks (time point 1, TP1) and 12 weeks (TP2) following the initiation of chemotherapy (Leukaemia and Lymphoma2002; 43: 1001). The aim of this study was to evaluate if polymerase chain reaction (PCR)-based MRD assay is sufficiently dependable for tailoring therapy, and if augmented therapy can reduce MRD levels to those associated with a favourable outcome. The subjects were under 18 years of age, and had newly diagnosed precursor B or T-cell ALL. Patients below one year old and those with t(9;22) were excluded. Written informed consent was obtained from patients or their legal guardians. The ALL 941-based protocol (45thASH, San Diego, 2003) utilized PCR-based MRD assay using immunoglobulin & T-cell receptor gene rearrangements. MRD was detected by nested PCR, with screening of rearrangements using multiplex PCR primers as described previously (Leukaemia and Lymphoma2002; 43: 1001). Patients were initially stratified into 3 risk groups (in ascending order: SR, HR, and HHR) according to leukocyte count and age at time of diagnosis. The MRD+/+ patients with levels ≥ 10−3 at both TP1 and TP2 received augmented therapy 14 weeks after initiation, and the remainder continued to receive the initial risk-adapted protocols. A total of 311 patients with a median age of 5.3 years (range 1.0–16.8) were eligible for this study. There were 4 (1.3%) non-responders and no deaths in induction. Of the 307 patients stratified, 169 (55%) were SR, 107 (35%) were HR, and 31 (10%) were HHR. The 2nd stratification by MRD level at TP2 was possible for 72.3% (222/307; insufficient DNA=28; missing time-points=25; no marker=32). Out of the 222 patients stratified, 125 (56.3%) were MRD−/−, 58 (26.1%) were MRD+/−, and 38 (17.4%) were MRD+/+. At the point of analysis, the median follow-up time was 63 months (range 33–89). The overall 5-year event–free survival (EFS) rate of the 307 patients was 80.1% (SE 2.5), higher than the EFS of the ALL941 study, which was 76.2% (SE 2.1) (p=0.167). The 5-year EFS rates according to the 1st stratification were 85.5% (SE 4) for SR, 76.1% (SE 4.5) for HR, and 64.6% (SE 9.2) for HHR, while the equivalent rates for the 2nd stratification were 87.0% (SE 3.1) for MRD−/−, 75.5% (SE 7.7) for MRD+/−, and 75.3% (SE 6.4) for MRD+/+. From the 95 patients whose MRD levels were measured at 5 consecutive points from TP1 to TP5 (5, 12, 18, 24, and 30 weeks after the start of therapy), 21 subjects with MRD+/+ received an augmented chemotherapy, and MRD levels became undetectable in 9 patients at TP3, 5 patients at TP4, and 4 patients at TP5. The corresponding cumulative 5-year relapse rates of those patients were 11%, 50%, and 50%, respectively. Thus, negative MRD status at TP3, but not at TP4 or TP5, seems to be associated with a favourable outcome. Our results confirm the strong performance of MRD-based treatment interaction in a multi-institutional study without adversely affecting the outcome in childhood ALL. Moreover, present findings suggest that an augmented therapy could reduce MRD to levels associated with a favourable outcome. To improve the applicability and accuracy of MRD assay, new MRD-PCR targets and RQ-PCR-based MRD detection are needed in subsequent studies. [Acknowledgment: This study was partly supported by grants from the Children’s Cancer Association of Japan (CCAJ)].

Description: Abstract 1112 Poster Board I-134 Background and objectives Although standard dose of IM, namely 400 mg daily, has been recommended for treating patients with chronic phase chronic myeloid leukemia (CML-CP), dose reduction is sometimes inevitable mainly because of adverse events. Previous reports have indicated that the trough plasma concentration (Cmin) of IM is a good indicator for predicting a good clinical response (Picard et al., Blood 2007; Larson et al., Blood 2008). Contrary, Forrest et al. reported that the Cmin correlates with neither cytogenetic nor molecular response (Leuk Res 2008). Therefore, it remains controversial whether the monitoring of IM Cmin is important and whether the dose adjustment should be based on Cmin. We thus assayed the Cmin in patients with CML-CP treated by standard or reduced doses of IM and analyzed their correlation with clinical response in a Japanese population. Methods Cmin samples were obtained 22 -26 hours after the last administration of IM from 46 patients with CML-CP treated for at least 12 months with imatinib at 7 hospitals in Ibaraki Prefecture, Japan. Cmin was determined using liquid chromatography-tandem mass spectrometry method. Clinical responses to IM, complete cytogenetic response (CCyR), major molecular response (MMR), and complete molecular response (CMR), were monitored simultaneously. CMR was defined as no detectable BCR-ABL transcripts by nested RT-PCR. Patient information was collected by questionnaire and medical chart review. Results Of evaluated 38 patients, 2 (5%), 6 (16%), 15 (39%), and 15 (39%) were treated with 100, 200, 300, and 400 mg of IM daily, respectively. The median duration of IM treatment was 1,777 days (range, 468-2799). The mean (±SD) and median Cmin in all patients were 979 ±599 and 836 ng/ml (range, 105-3,700 ng/ml), respectively. The mean (±SD) Cmin at dose of 100, 200, 300, and 400 mg/day were 210±148, 715±209, 940±394, and 1,226±771 ng/ml, respectively. The Cmin was significantly higher in patients treated with standard dose than those treated with the reduced dose (P= 0.04). A total of 33(86.8%) patients achieved a CCyR. There was no significant difference in the mean Cmin (±SD) between patients who achieved CCyR and who did not (977±605 vs 993±631 ng/ml, P=0.48). No significant difference was observed in the IM daily doses (P=0.17) and in the duration of IM treatment (P=0.96) between patients with and without CCyR. One (50%), 4 (50%), 9 (60%), and 13 (87%) patients treated at IM dose levels of 100, 200, 300, 400 mg/day achieved MMR, respectively. However, there was no significant difference in the IM daily dose (P=0.37) and in the duration of IM treatment (P=0.35) between patients with and without MMR. The mean IM Cmin (±SD) of patients with MMR was 1,044±635 ng/ml, which was not significantly higher than those without MMR (818±488 ng/ml, P=0.17). These data collectively suggest that neither IM Cmin nor daily dose is relevant for achieving CCyR and MMR in our population. However, importantly, 7 of 15 patients treated with 400 mg/day of IM achieved CMR, whereas no patients treated with 100 or 200 mg/day and only one patient with 300 mg/day achieved CMR. Indeed there was significant difference in the daily dose of IM between patients with and without CMR (P=0.02). The mean Cmin (±SD) with CMR was 1,430±988 ng/ml, which was significantly higher than those without CMR (859±389 ng/ml, P=0.04). Contrary to this, Cmin of patients treated with 400 mg/day was not significantly different between patients with and without CMR (P=0.22). The duration of IM treatment was not different between patients with and without CMR (P=0.75). Finally, in multivariate analyses using a logistic regression model, the estimated odds ratio of achieving CMR for patients treated with standard versus reduced dose of IM was 22.1 (95% CI 1.5-330.5). There was no correlation of CMR and age, sex, body surface area, or the IM Cmin. Conclusions/Methods We believe that adherence to the standard dose of IM have a greater impact on achieving deep molecular response than adjusting its therapeutic dose based on Cmin in practical clinics. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: With current intensified chemotherapy, more than 70% of children with acute lymphoblastic leukemia (ALL) are curable. However, prognosis of the patients at higher risk has been unsatisfied. More appropriate risk assignment and innovative treatment is expected to be developed. We conducted a clinical trial of JACLS ALL-97 including multi-agent block therapy followed by hematopoietic stem cell transplantation (SCT) for the selected higher risk patients. PATIENTS AND METHODS: Between April 1997 and March 2002, 674 patients aged 1 to 15 years with newly diagnosed ALL (excluding mature B-cell ALL) were enrolled on the JACLS ALL-97 protocol. Excluding 75 T-ALL, 26 mixed lineage leukemia other than B-precursor ALL with myeloid markers, 9 acute unclassified leukemia and 1 B-precursor ALL with pretreatment, 563 patients with B-precursor ALL were eligible for this analysis. Treatment group was divided 5 groups according to the modified National Cancer Institute (NCI) workshop criteria. Standard risk (SR) and IR (intermediate risk) divided by WBC10 × 103 account for NCI-SR. High risk (HR) and ER (extremely high risk) divided by WBC 100 × 103 account for NCI-HR. The patients with ALL positive for Philadelphia chromosome and for translocation with chromosome11q23 were assigned to the highest risk group F. The patients in complete remission (CR) at day 35 with M2/M3 at the day 14 marrow were assigned to shift higher risk after induction therapy. Treatment of ALL-97 consists of early phase and maintenance phase. Early phase includes induction therapy, consolidation therapy, sanctuary therapy, and re-induction therapy for 19 to 26 weeks dependent on risk group. Early phase in IR/HR protocol is identical to the SR protocol except the addition of two doses of DNR and three doses of CPM. Maintenance phase contains standard MTX and MP with monthly VCR and PSL intensification for SR, and rotational therapy of a set of MTX and MP with a set of VCR, PSL, ASP, and (DNR or CPM) for IR/HR/ER/F. The IR protocol is identical to the HR protocol without cranial irradiation. Treatment duration is 24 months for any risk. The patients assigned ER and F were candidate for allogeneic stem cell transplantation by the end of early phase. Transplant procedures depended on the institute. RESULTS: Number of patients at each risk was 204 for SR, 158 for IR, 128 for HR, 36 for ER, 27 for Ph+ and 10 for 11q23. Six of them were treated with incorrect risk protocol. Thirty-four patients received SCT in first CR. Following the induction therapy, 550 of 564 patients (97.5%) achieved CR. 23 patients(8 in SR, 9 in IR, 4 in HR and 2 in ER, 4.4% of all) were shift to higher risk due to the findings of day 14 marrow. Five-year overall survival rate (OS) and event-free survival rate (EFS) for all patients was 90.6% and 77.0%, respectively. Five-year EFS for NCI-SR and HR was 81.6% and 67.6%, respectively. According to risk group, 5-year EFS for SR, IR, HR, ER, Ph+ ALL, and ALL with 11q23 were 86.6%, 77.0%, 71.9%, 68.7%, 40.7%, and 70.0%, respectively. 5-year OS for them were 94.0%, 93.9%, 92.1%, 83.8%, 55.6%, and 68.6%, respectively. CONCLUSIONS: By the risk-adapted therapy in the JACLS ALL-97 trial, high cure rate could be achieved for children with B-precursor ALL.

Description: With modern risk-adapted multi-agent chemotherapy, complete remission (CR) is obtained in the majority of children with acute lymphoblastic leukemia (ALL) and long-term survival free from disease can be expected in more than 70% of them. However, the prognosis of the patients without CR (induction failure; IF) has been dismal. We conducted a prospective study of F-protocol followed by a hematopoietic stem cell transplantation (SCT) in patients with IF. Purpose: To evaluate the efficacy and safety of the treatment strategy adopted in the JACLS ALL F-protocol. Patients and methods: Between April 1997 and March 2005, 1,254 patients with newly diagnosed ALL (excluding mature B-cell ALL) were enrolled on two consecutive JACLS ALL protocols (ALL-97 and ALL-02). Excluding induction death, 19 (1.6%) out of 1,207 patients with non-Ph+ ALL and 9 (30%) out of 30 patients with Ph+ ALL could not achieve CR following four- or five-drug induction chemotherapy. Among them, 25 patients in total were entered the study with guardian’s written consent. F-protocol consisted of the AML oriented re-induction chemotherapy (3 days of 8 mg/m2 MIT, 500 mg/m2 CA, and 40 mg/m2 PSL, followed by 3 days of 200 mg/m2 VP16, CA, and PSL a week later), four block consolidation therapy consisting of two alternative regimens A and B (A: HD-DEX, VP16, CA, MIT, B: HD-MTX, ASP, PSL, CA, THP-ADR) and maintenance therapy. Patients with CR were scheduled to receive SCT after the second consolidation therapy before the maintenance therapy. Transplant procedures depended on the institute. Results: Following the re-induction therapy, 15 of 16 patients with non-Ph+ ALL (93.8%) achieved CR and 13 (86.7%) of them had continued CR until 27th week of treatment (CCR), which was the expected limiting time of SCT. Eight of 11 patients receiving SCT at the first CCR are alive. Two of 13 patients with CCR did not receive SCT by a physician’s decision, and relapsed during maintenance therapy. However they are alive following SCT in second CR. Five-year overall survival rate (OS) and event free survival rate for all 16 cases was 53% and 32.8%, respectively. Estimated five-year OS for patients receiving SCT at the first CCR was 59.7%. On the other hand, only 3 of 9 patients with Ph+ ALL (33.3%) achieved CR. No patients without response to re-induction therapy could achieve CR by any subsequent chemotherapy, but one success with SCT. Among 3 patients attaining a CR with re-induction therapy, 2 could continue CR and underwent SCT. One is alive in CCR and the other deceased from TRM. The other patient, relapsed during consolidation therapy, underwent SCT with success. Five-year OS for all 9 patients with Ph+ ALL was 33.3%. Concerning about adverse events of F-protocol, there was only one non-hematological toxicity NCI-CTC grade 4, which was pancreatitis due to ASP. Conclusions: F-protocol could produce a high remission induction rate in non-Ph+ ALL, but not in Ph+ ALL. Improved outcome of non-Ph+ ALL patients with IF seemed to be obtained with F-protocol timely followed by SCT.

Description: Angiogenesis, an essential process for tumor growth, is regulated by endothelial proliferation factors and their inhibitors such as endostatin. Endostatin, a carboxyl-terminal fragment of type XVIII collagen, inhibits endothelial proliferation, angiogenesis, and tumor growth. Ornithine decarboxylase (ODC), a molecule that is overexpressed in various cancers, is associated with promoting tumor growth and angiogenesis. We found that ODC-overexpressing human cancer cells and breast cancer specimens showed suppressed expression of type XVIII collagen and endostatin. We hypothesized that ODC overexpression may facilitate angiogenesis in tumors by suppressing endostatin expression. ODC-overexpressing COS cells, which showed suppressed type XVIII collagen and endostatin expression, were established. Conditioned media derived from these cells, containing decreased levels of endostatin, induced significant endothelial proliferation. ODC-overexpressing cells, when transplanted into nude mice, suppressed type XVIII collagen expression and promoted neovascularization in vivo. Thus, overexpression of ODC facilitates endothelial proliferation by suppressing endostatin expression.

Description: ErbB2, c-Myc and p53 gene expressions are widely reported for the poor prognostic markers for medulloblastoma (MB) patients. Recently, Wnt pathway and Sonic Hedgehog (SHH) pathways are known to related to the oncogenesis of MB cells, and the PDGF receptor gene and some adhesion molecule gene expressions are also known to the markers of the dissemination or the metastasis of MB cells. For adult malignant brain tumors, the molecular targeted therapy of the antibody (trastuzumab, cetuximab), the tyrosine kinase inhibitor (imatinib, gefinitib) or other signal transduction inhibitor (cyclopamin) are clinically researched. We examined several important marker gene expressions of the molecular therapy in various children’s brain tumors and searched the possible molecular targeted therapy. [Materials and methods] Fifteen children’s brain tumor (five MB, seven glioma, three ependymoma) and four adult brain tumor (two glioma, two glioblastoma) were examind. The three MB cases were primary metaststic ones. The mRNA expressions of the marker genes (ErbB2, PDGF receptor, PCNA, SPARC, β-catenin, SUFU, c-Myc, p53, TrkC and so on) were examined by quantity polymerase chain (qPCR) reaction with fresh frozen tumor cells. For the normal control, we used the normal cerebellum total RNA samples of the Becton, Dikinson and Company. CYBR green coloring system was used for the qPCR and their primers were designed in the region between two exons. GAPDH gene expression was also examined as an internal control and all of the gene expression were normalized by GAPDH expression. [Results] ErbB2 gene expression in MB cells were various, but their expression were similar to clinical futures, such as, the higher expressed cases had some high risk factors. In glioma cells, ErbB2 gene expression were almost equal by lower levels. PDGF receptor gene expression were more than five to ten times elevated in all of the glioma cells, even if they were low grade malignancy glioma, and were higher than that in MB cells. PCNA was specially elevated in primary metastatic MB cells. [Conclusion] Our data shows that the molecular characteristics of the children’s brain tumors were thought to be different by cases, even if they had same histopathological characters. Some special gene tageting therapy, such as anti-ErbB2, PDGFR and/or PCNA therapy can be used for the various children’s brain tumors which are resistant for conventional therapies and are cured by adequate molecular therapy.

Description: Persons who were treated mainly in 80’s were reviewed and assessed for the present status including late effects, school, social life, marital state. Questionaire was sent to individual institutions and answers were returned from physicians or survivors themselves. From 1881 to 1994, there were five clinical protocols in ALL, four in AML, and three in NHL. Registered and surviving numbers were 1,023 and 642 in ALL, 309 and 164 in AML, 200 and 166 in NHL. Of surviving numbers, responded number was 521 in ALL, 142 in AML, 103 in NHL. Of these 1,262 survivors, 128 persons have proven to have some kinds of late effects. Mean age of survivors was 6.7 at the onset of disease and 18.6 at the time of this survey. Questionair was as followings; diagnosis, gender, age, presence or absence of late effects, detail of late effects, and present status. As a result, 128 survivors (male/female; 78/50, ALL; 75, AML; 30, NHL; 23) have 169 items of late effects. The overall incidence of late effects was 13% in ALL and 20% in AML and NHL. There was no difference of incidence between each treatment protocols in ALL. Additionally, 10 persons died with late effects. Of those, the cause of death was as followings; leukoencephalopathy; 3, cardiac toxicity 3, of them, one boy was in remission after completion of chemotherapy and died during running, relapse of primary disease; 1, secondary cancer; 1, measles; one who was in remission at the time of infection, ARDS; one who received stem cell transplantation. The late effects were classified as following categories (sALL/hALL/AML/NHL); hepatic 30(14/7/6/3), cardiac 7 (3/2/0/2), central nervous system 33 (13/6/6/8), short stature 29 (14/6/5/4), gonad 16 (3/4/9/0), thyroid 4 (1/1/2/0), renal 8 (3/1/2/2), pulmonary 6 (1/2/2/1), cGVHD 4 (1/2/1/0), secondary cancer 6 (4/0/0/2), others 26 (5/3/11/7). The number of each category of late effects was so small, that decreasing tendency was not apparent with time course of this studying period except hepatic disorder, which is related to post-transfusion hepatitis C. CNS disorders include leukoencephalopathy, mental retardation, epilepsy, and visual disturbances. More than one half of short stature was found in the standard risk ALL. Most of gonad dysfunction, persistent alopecia, pulmonary and renal disorders were related to stem cell transplantation. Psychological or mental disturbance, which are related school troubles, was also observed. Second cancer found in six persons. Primary diagnosis of secondary cancer was ALL in 4 and NHL in 2. For these persons who have late effects, present state of individual life was evaluated and classified into four categories; (1) usual life without handicapped and medical service, (2) no handicapped, but necessary medical service, (3) handicapped, but unnecessary medical service, (4) handicapped, and necessary medical service. Each category was found in any primary disease or type of treatment. Discussion: The several major late effects of survivors will not decrease in near future, considering the number of survivors is increasing for these decades. Novel system for survivors should be build up as to make their lives healthier.

Description: Unlike conventional apoptosis, the mutually exclusive role of ceramide and sphingosine-1-phosphate (S1P) in autophagic cell death remains unclear in leukemia cells. Amino acids deprivation (AA(−))-induced autophagy and subsequent cell death of human leukemia HL-60 cells are preceded by the generation of intracellular ceramide with inhibition of mammalian target of rapamycin (mTOR) in a caspase-3-independent manner. S1P inhibits AA(−)- or N-acetylsphingosine (C2-ceramide)-induced autophagy through activation of an amino acids sensor mTOR, whereas C2-ceramide inhibits S1P-activated mTOR and overcomes inhibition of autophagy by S1P. Genetically activated mTOR inhibits AA(−)- or C2-ceramide-induced increases of autophagy and a mammalian homologue of yeast Apg8/Aut7, MAP-LC3. In contrast, overexpression of kinase-dead mTOR blocks S1P-induced inhibition of autophagy and LC3-II activation. We here suggest that in leukemia cells ceramide an S1P counteract as a novel mediator for autophagic cell death through regulation of mTOR-dependent MAP-LC3.

Description: [Objective]Adult T cell leukemia (ATL) is extremely resistant to conventional chemotherapy and hard to cure even with allogeneic stem cell transplantation. Cytotoxic T-lymphocyte-based immunotherapy is expected to be one the therapeutic options for ATL in the near future. So far, however, there are no promising leukemia-associated antigens for immunotherapy of ATL. It is known that most leukemic cells of ATL in vivo hardly express HTLV-I-associated viral antigens. Furthermore, in contrast to solid tumors, only a few tumor (leukemia)-associated antigens have been identified in hematological malignancies. Thus, it is crucial for establishment of immunotherapy of ATL to identify novel applicable leukemia-associated antigens. In the present study, we attempted a comprehensive analysis of MHC class I peptides presented on ATL cells by the latest technology of mass-spectrometry (MS) which has recently made rapid progress. [Materials&Methods]Three ATL-derived cell lines, ED-D40515, ATL-43T, and ATL-55T+ were cultured and expanded on a large scale, each of which has been demonstrated to be clonally identical to original leukemic cells by southern blot analysis of either HTLV-I provirus or TCR rearangement. Cell lysates prepared from 2–3×109 cells were incubated with Affigel-Hz (Bio-Rad) coupled with anti-HLA class I mAb (W6/32). Affigel resin beads were washed extensively and then subjected to elution with acidic buffer. MS analyses were performed using a LCQ Deca ion trap instrument (ThermoQuest) equipped with a directly communicated on-line reversed- phase high performance liquid chromatography (RP-HPLC) system (MichromBioResources). Protein identification was performed using the SEQUEST algorithm maintained at the National Center for Biotechnology Information (NCBI). We analyzed MHC class I restriction of the candidate peptides and assorted them based on the algorithm of HLA ligands databases SYFPEITHI. [Results]We were able to sequence approximately 60 peptides per cell line which were derived from different source proteins and presented by respective HLA class I alleles. Although most peptides were derived from ubiquitous or constitutively expressed proteins in normal CD4+ T cells, 10 source proteins that we identified had been previously reported as the cancer-testis antigens or the cancer-associated and/or -overexpressed gene products. We are currently screening most peptides including those derived from unknown gene products by comparing mRNA expression in ATL cells and normal tissues. Up to now, 7 candidate peptides have been identified that seem to be derived from leukemia-associated antigens specifically expressed in ATL cells. [Discussion]Recent progress in proteomics technology has enabled us to directly determine sequences of numerous peptides bound to MHC molecules. In the present study, we demonstrated that LC-MS is a feasible and efficient method to analyze comprehensive repertoire of MHC class I peptides presented on ATL cells. Several candidate peptides presumably derived from leukemia-associated or -overexpressed antigens were identified based on MHC class I restriction and mRNA expression pattern in leukemic cells and normal tissues. It is to be tested whether these peptides can induce bonafide CTL response against ATL cells and at the end be applicable to immunotherapy of ATL. Considering that expression patterns of leukemia-associated antigens vary from case to case, this approach appears to be suitable for the tailor-made immunotherapy.