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Background Histological images show strong variance (e.g. illumination, color, staining quality) due to differences in image acquisition, tissue processing, staining, etc. This can impede downstream image analysis such as staining intensity evaluation or classification. Methods to reduce these variances are called image normalization techniques. Methods In this paper, we investigate the potential of CycleGAN (cycle consistent Generative Adversarial Network) for color normalization in hematoxylin-eosin stained histological images using daily clinical data with consideration of the variability of internal staining protocol variations. The network consists of a generator network GB that learns to map an image X from a source domain A to a target domain B, i.e. GB:XA→XB. In addition, a discriminator network DB is trained to distinguish whether an image from domain B is real or generated. The same process is applied to another generator-discriminator pair (GA,DA), for the inverse mapping GA:XB→XA. Cycle consistency ensures that a generated image is close to its original when being mapped backwards (GA(GB(XA))≈XA and vice versa). We validate the CycleGAN approach on a breast cancer challenge and a follicular thyroid carcinoma data set for various stain variations. We evaluate the quality of the generated images compared to the original images using similarity measures. In addition, we apply stain normalization on pathological lymph node data from our institute and test the gain from normalization on a ResNet classifier pre-trained on the Camelyon16 data set. Results Qualitative results of the images generated by our network are compared to original color distributions. Our evaluation indicates that by mapping images to a target domain, the similarity training images from that domain improves up to 96%. We also achieve a high cycle consistency for the generator networks by obtaining similarity indices greater than 0.9. When applying the CycleGAN normalization to HE-stain images from our institute the kappa-value of the ResNet-model that is only trained on Camelyon16 data is increased more than 50%. Conclusions CycleGANs have proven to efficiently normalize HE-stained images. The approach compensates for deviations resulting from image acquisition (e.g. different scanning devices) as well as from tissue staining (e.g. different staining protocols), and thus overcomes the staining variations in images from various institutions.The code is publicly available at https://github.com/m4ln/stainTransfer_CycleGAN_pytorch. The data set supporting the solutions is available at 10.11588/data/8LKEZF.

Background For the histopathological diagnosis of lymphoma, lymph node excision biopsies are regarded as standard of care. In contrast, for the diagnosis of carcinoma and deep-seated tumors, core needle biopsies (CNBs) are accepted as a sufficient sampling method. We evaluated a diagnostic algorithm for peripheral lymphadenopathy starting with ultrasound-guided CNB followed by excisional biopsy in ambiguous cases. Methods We performed ultrasound-guided CNB of peripheral lymph nodes and subcutaneous tumors in patients with lymphadenopathy in routine care and analyzed its accuracy, complication rate, and the impact of needle sizes on results. Results From 138 patients, 132 samples were technically adequate. In 121 patients, CNB provided a clinically actionable diagnosis (76 lymphoma, 30 carcinoma, 15 non-malignant diagnoses). A total of 54 patients had a secondary biopsy. Inconclusive diagnoses were rare with two false-positive and two false-negative non-Hodgkin's lymphoma, and higher for Hodgkin's lymphoma with five false-negative cases. The rate of complications was low. Needle size did not significantly influence results. Conclusion Ultrasound-guided CNBs are a safe, quick, and valid tool for the workup of lymphadenopathy. Yet, a benign diagnosis from CNB must be completed by a secondary biopsy if clinical presentation suggests malignant disease.

Toll-like receptor 9 (TLR9) agonists, commonly referred to as CpG oligodeoxynucleotides (ODN), have been added to the arsenal of anti-cancer drugs as monotherapy or in combination with chemotherapy, radiotherapy and other immunotherapeutic approaches as they increase antigen presentation and boost anti-tumor T- and B-cell responses. Several synthetic TLR9 agonists have been developed for clinical grade use and displayed substantial efficacy in the preclinical and clinical models.This review summarizes TLR9 signaling and the impact of TLR9 agonists on the immune response. The most recent experimental and clinical data are analyzed as well as the development of new TLR9 agonists in current clinical trials.Application of TLR9 agonists, in particular, combination strategies with chemo- or radiotherapy seem a promising and efficient immunotherapeutic approach in cancer patients even with refractory disease. Simultaneous application of TLR9 agonists aims at supporting the patient's immune response and overcoming specific immunosuppressant strategies developed by tumors. Combinatory approaches of the future might also seek for synergism of TLR9 agonists with other immunomodulatory strategies such as B-cell activation using the CD40-CD40L system.

Background: The combination of chemotherapy with the vascular endothelial growth factor (VEGF) antibody bevacizumab is a standard of care in advanced colorectal cancer (CRC). However, biomarkers predicting outcome of bevacizumab-containing treatment are lacking. As angiopoietin-2 (Ang-2) is a key regulator of vascular remodelling in concert with VEGF, we investigated its role as a biomarker in metastatic CRC. Methods: Serum Ang-2 levels were measured in 33 healthy volunteers and 90 patients with CRC. Of these, 34 had metastatic disease and received bevacizumab-containing therapy. To determine the tissue of origin of Ang-2, quantitative real-time PCR was performed on microdissected cryosections of human CRC and in a murine xenograft model of CRC using species-specific amplification. Results: Ang-2 originated from the stromal compartment of CRC tissues. Serum Ang-2 levels were significantly elevated in patients with metastatic CRC compared with healthy controls. Amongst patients receiving bevacizumab-containing treatment, low pre-therapeutic serum Ang-2 levels were associated with a significant better response rate (82 vs 31% P

Purpose: Cyclin D1, a key cell cycle regulator, is overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for cancer immunotherapy. Nevertheless, it had previously been suggested that efficient T cells recognizing cyclin D1-derived epitopes are absent from the repertoire because of thymic deletion. We attempted to induce autologous CTL from healthy donors and patients with cyclin D1-overexpressing tumors using a highly efficient T-cell expansion system based on CD40-activated B cells as antigen-presenting cells.Experimental Design: Cyclin D1-derived, HLA-A*0201–restricted epitopes were predicted by multiple computer algorithms, screened in HLA-A2-binding assays, and used for T-cell stimulation. The generated CTL lines and clones were analyzed by IFN-γ enzyme-linked immunosorbent spot assay or cytolysis assay.Results: After screening, at least two naturally processed and presented HLA-A*0201–binding cyclin D1 epitopes were identified. CTL specific for these epitopes could be successfully generated from HLA-A2+ donors. T cells efficiently recognized target cells pulsed with the cognate peptide and cyclin D1-expressing tumor cell lines in an HLA-A*0201–restricted manner. More importantly, HLA-A*0201–matched, primary cyclin D1+ tumor cells were efficiently recognized by cyclin D1-specific CTL. These CTL could be generated from patients with mantle cell lymphoma and cyclin D1+ colon cancer.Conclusions: These results underscore that cyclin D1 needs to be considered as a target for broad-based antitumor immunotherapy.

Stage IV colorectal cancer is usually an incurable disease. However, patients with resectable metastases have 5-year disease-free survival rates of up to 30%. Even with primarily irresectable disease, cure can be achieved in patients who become operable after neoadjuvant treatment. To improve the prognosis of these patients, highly effective neoadjuvant regimens need to be developed.Here, we report the case of a 62-year-old male patient who had been diagnosed with International Union against Cancer (UICC) stage III colon cancer 7 years previously and now presented with a large, irresectable liver metastasis and enlarged perihepatic lymph nodes. After neoadjuvant treatment with cetuximab, bevacizumab and XELOX, the patient showed a complete remission and underwent surgery. Histopathologically, the resected tissue and lymph nodes were free of residual tumor.To our knowledge, this is the first report of a complete pathological response in a patient with irresectable colorectal cancer after intensive chemotherapy/anti-EGFR/ VEGF antibody therapy. This combination regimen may help to improve the survival rates for patients with irresectable disease.

Purpose: We conducted a phase I/II randomized trial to evaluate the clinical and immunologic effect of chemotherapy combined with vaccination in primary metastatic colorectal cancer patients with a carcinoembryonic antigen–derived peptide in the setting of adjuvants granulocyte macrophage colony-stimulating factor, CpG-containing DNA molecules (dSLIM), and dendritic cells. Experimental Design: HLA-A2–positive patients with confirmed newly diagnosed metastatic colorectal cancer and elevated serum carcinoembryonic antigen (CEA) were randomized to receive three cycles of standard chemotherapy (irinotecan/high-dose 5-fluorouracil/leucovorin) and vaccinations with CEA-derived CAP-1 peptide admixed with different adjuvants [CAP-1/granulocyte macrophage colony-stimulating factor/interleukin-2 (IL-2), CAP-1/dSLIM/IL-2, and CAP-1/IL-2]. After completion of chemotherapy, patients received weekly vaccinations until progression of disease. Immune assessment was done at baseline and after three cycles of combined chemoimmunotherapy. HLA-A2 tetramers complexed with the peptides CAP-1, human T-cell lymphotrophic virus type I TAX, cytomegalovirus (CMV) pp65, and EBV BMLF-1 were used for phenotypic immune assessment. IFN-γ intracellular cytokine assays were done to evaluate CTL reactivity. Results: Seventeen metastatic patients were recruited, of whom 12 completed three cycles. Therapy resulted in five complete response, one partial response, five stable disease, and six progressive disease. Six grade 1 local skin reactions and one mild systemic reaction to vaccination treatment were observed. Overall survival after a median observation time of 29 months was 17 months with a survival rate of 35% (6 of 17) at that time. Eight patients (47%) showed elevation of CAP-1–specific CTLs. Neither of the adjuvants provided superiority in eliciting CAP-1–specific immune responses. During three cycles of chemotherapy, EBV/CMV recall antigen–specific CD8+ cells decreased by an average 14%. Conclusions: The presented chemoimmunotherapy is a feasible and safe combination therapy with clinical and immunologic efficacy. Despite concurrent chemotherapy, increases in CAP-1–specific T cells were observed in 47% of patients after vaccination.

Today, many patients with lymphoma are cured by polychemotherapy and irradiation. However, residual masses are frequently observed after treatment and discrimination between vital tumor and inactive fibrotic tissue by computed tomography or magnetic resonance tomography is often not possible. 18 F-Fluorodeoxyglucose (FDG) positron emission tomography (PET) is a metabolic imaging modality that is able to detect active lymphoma lesions. The application of PET may play a crucial role in identifying patients with residual disease and contribute valuable prognostic information. To assess the prognostic implications of PET in the post-therapeutic setting, we performed a MedLine Search and reviewed the current available studies on this important issue together with our own data.

PURPOSE: Micrometastatic disease in bone marrow is of prognostic significance in colorectal cancer patients. However, detection rates of standard immunocytology are relatively low. We used magnetic activated cell sorting (MACS), a highly sensitive method, to increase detection rates and correlated the presence of cytokeratin (CK)-expressing cells with clinical parameters. PATIENTS AND METHODS: Bone marrow was obtained from 51 consecutive patients with newly diagnosed colorectal adenocarcinoma who underwent primary surgery and 18 control subjects. International Union Against Cancer (UICC) stage I disease was diagnosed in 11 patients, stage II disease was diagnosed in 14 patients, stage III disease was diagnosed in 12 patients, and stage IV disease was diagnosed in 14 patients. CK-positive cells were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to CK 7 and 8. RESULTS: CK-positive cells were found in 33 (65%) patients and were absent in 18 (35%). Four of 11 (36%) patients with UICC stage I disease, nine of 14 (64%) with stage II diease, eight of 12 (67%) with stage III disease, and 12 of 14 (86%) with stage IV disease were CK-positive. Epithelial cells were more frequently found in pT3/4 (72%) than in pT1/2 (36%) tumors (P = .026), but there was no difference for lymph node status. CK-positive patients had a higher chance for elevated carcinoembryonic antigen (85% v 15%, P = NS) and CA 19-9 levels (92% v 8%, P = .019). There were no significant differences in CA 72-4, sex, age, tumor grading, or tumor localization regarding the presence of CK-positive cells. All control subjects were CK-negative. CONCLUSION: In searching for micrometastases in colorectal cancer patients, we have achieved high detection rates by using MACS. The presence of these cells correlated significantly with tumor stage, tumor extension, and the tumor marker CA 19-9.

High-dose methotrexate (HDMTX) is a component of many cancer treatment regimens. Despite careful management, delayed renal clearance, followed by extremely high serum levels with potentially life-threatening toxicity can occur. In the present study, we report our results of carboxypeptidase-G2 (CPDG2) rescue in 8 patients with delayed methotrexate elimination and renal impairment after HDMTX therapy for lymphoma or osteosarcoma. A dose of 50 U/kg CPDG2 was administered. MTX plasma levels decreased rapidly and recovery of renal function was observed in all patients. No patient developed severe WHO grade 4 MTX toxicity. CPDG2 provides an alternative route of MTX elimination by converting it to inactive and non-toxic metabolites. CPDG2 rescue was well tolerated, safe and very effective in preventing severe or life-threatening MTX toxicity.

The detection of isolated tumor cells (ITC) in the bone marrow of patients with epithelial malignancies is an independant prognostic factor for several entities as breast cancer, colorectal cancer or non-small lung cancer. However, with conventional immunocytology using Ficoll density gradient and APAAP staining, only a small proportion of the bone marrow samples can be scanned for cytokeratin-positive (CK+) cells. To improve detection rates, we evaluated the enrichment of ITC by magnetic activated cell sorting (MACS) compared to regularly stained cytospins. Recovery experiments with a CK+ breast cancer cell line (SKBR3) were performed to calculate the MACS enrichment rate. Bone marrow was obtained by aspiration from 20 patients with carcinomas of epithelial origin and from 17 controls. ITC were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to cytokeratin 7 and 8. MACS of SKBR3 seeded in peripheral blood revealed average recovery rates of 62% and 48% and average enrichment factors of 104-fold and 8139-fold of the CK+ cells after one and after two separations, respectively. After immunomagnetic enrichment, CK+ cells were detected in 16 of 20 (80%) cancer patients, whereas only 7 (35%) patients showed CK+ cells without magnetic enrichment (P=0.002). Ten of twelve (83%) patients with metastatic disease (stage M1) and six of eight (75%) patients without any overt metastases (M0) had CK+ cells in their bone marrow. None of the negative controls showed any CK+ cells. Enrichment with magnetically labeled anti cytokeratin antibodies increases the detection rate of epithelial cells in bone marrow of cancer patients compared to immunocytology.

Hintergrund: Kunstliche Susstoffe haben in den letzten 20 Jahren eine rasante Entwicklung durchgemacht und werden heutzutage einer breiten Palette von Getranken, Speisen, Medikamenten und Hygieneartikeln beigefugt. Seit ihrer Einfuhrung sind in den Medien immer wieder Meldungen uber mogliche Krebsrisiken zu vernehmen, die zu einer Verunsicherung der Bevolkerung fuhren. Man kann davon ausgehen, dass heutzutage jeder Burger der westlichen Lander kunstliche Susstoffe, wenn auch teilweise unbewusst, verwendet. Eine mogliche krebserregende Wirkung dieser Substanzen hatte daher Auswirkungen auf die Bevolkerungsgesundheit. Studien: Die Arbeit gibt eine Ubersicht zu den wichtigsten publizierten Studien, die das kanzerogene Potential von kunstlichen Susstoffen untersuchen.

HISTORY AND FINDINGS A 38-year-old female physician was admitted with the suspected diagnosis of a myelodysplastic syndrome. The self-reported history was inconsistent and incoherent. On physical examination neurological and dermatological disorders could be found. EXAMINATIONS A bone marrow puncture revealed a toxicity-induced bone marrow damage. Extensive examinations to evaluate the neurological symptoms did not show any pathological findings. A toxicological screening identified opioids, benzodiazepines and promethazine. TREATMENT AND COURSE Because of the inconsistent history and the contradictory results of our clinical tests, we asked in other hospitals for past admissions and were thus able to have insight into a hospital career that grasped numerous different admission diagnoses. All presented symptoms, especially the bone marrow damage, were self-induced by the patient. CONCLUSIONS For the first time in this patient's history, a factitious disorder was diagnosed after a hospital career of almost 10 years. In this publication, the case is thoroughly presented. Additionally, the current literature is reviewed with latest recommendations for therapeutic strategies about this poorly understood disorder.

Tie2-expressing monocytes (TEM) promote tumor angiogenesis and growth in experimental cancer models. The role of TEM in cancer patients is unknown. We studied TEM in healthy volunteers and colorectal cancer (CRC) patients. Although TEM were detectable in the blood and tumor lesions of CRC patients, their frequency and functional phenotype showed no correlation with levels of angiopoietin-2 or vascular endothelial growth factor, microvessel density, tumor markers, tumor stage, or outcome of antiangiogenic therapy. These unexpected findings are at odds with murine tumor models and question the diagnostic or therapeutic value of TEM in human cancer.

In recent years an increase of functional CD4+CD25+regulatory T cells (Tregcells) has been established for patients with solid tumors, acute leukemias, and lymphomas. We have reported an expanded pool of CD4+CD25highTregcells in patients with chronic lymphatic leukemia (CLL), multiple myeloma (MM) as well as its premalignant precursor monoclonal gammopathy of undetermined significance (MGUS). In healthy individuals, low-level expression of CD127 on T cells in addition to the expression of FOXP3 has been associated with Tregcells. Here, we demonstrate that the expanded FOXP3+T-cell population in patients with colorectal cancer, CLL, MGUS, MM, follicular lymphoma, and Hodgkin's disease are exclusively CD127lowTregcells and were strongly suppressive. A significant portion of CD127lowFOXP3+Tregcells expressed only low levels of CD25 suggesting that the previously reported expansion of CD25+Tregcells underestimates the true expansion. The assessment of CCR7 and CD45RA expression on the expanded CD4+CD127lowFOXP3+Tregcells revealed an increase of both naïve as well as central and effector memory Tregcells in peripheral blood. Our data strongly support superiority of combined CD127 and FOXP3 analysis in comparison to CD25 and FOXP3 assessment for further quantification of Tregcells in malignant diseases.

Evaluation of: Brody JD, Ai WZ, Czerwinski DK et al. In situ vaccination with a TLR9 agonist induces systemic lymphoma regression: a Phase I/II study. J. Clin. Oncol. 28(28), 4324-4332 (2010). Despite high response rates of the follicular B-cell lymphoma to monoclonal antibodies, the clinical course of lymphoma is still characterized by a continuous pattern of relapse. Brody and colleagues treated 15 patients with relapsed, low-grade lymphoma using low-dose radiotherapy applied to one of the tumor sites with combined injection of a TLR-9 agonist at the same site. This strategy induced specific immunity and tumor regression in several patients with an objective response rate of 27%. The results indicate an involvement of the tumor TLR-9-expressing B cells acting as antigen-presenting cells. TLR-9 in situ vaccination combined with local radiotherapy clearly warrants further in-depth analysis and investigation in a Phase III randomized trial, and may provide a new opportunity for the treatment of B-cell malignancies.

Background Sunitinib monotherapy in pretreated patients with advanced gastric cancer (AGC) was investigated. Preplanned analyses of tumour biomarkers on treatment outcome were performed. Patients and methods Patients received sunitinib 50 mg/day for 4 weeks with 2 weeks rest until disease progression or unacceptable toxicity. The primary end-point was objective response rate (ORR). Secondary end-points included progression-free survival (PFS), overall survival (OS) and safety. Results Fifty-two patients were enrolled and treated (safety population, SP). In the intention to treat population ( n = 51); the ORR was 3.9%, median PFS was 1.28 months [95% CI, 1.18–1.90], median OS was 5.81 months [95% CI, 3.48–12.32], the estimated one-year survival rate was 23.7% [95%CI: 12.8–36.5]. In subgroup analyses, tumour VEGF-C expression compared with no expression was associated with significantly shorter median PFS (1.23 versus 2.86 months, logrank p = 0.0119) but there was no difference in tumour control rate ( p = 0.142). In the SP, serious adverse events occurred in 26 patients, leading to 13 deaths, all sunitinib unrelated. Thirty-eight patients died from progressive disease, nine died Conclusion Sunitinib monotherapy was associated with limited tumour response and good/moderate tolerability in this setting.

SummaryCellular adjuvants such as dendritic cells (DC) are in the focus of tumour immunotherapy. In DC-vaccine trials, induction of tumour antigen-specific immunity is observed frequently and well-documented clinical responses have been reported. However, the overall response rate is less than 3%, therefore alternative strategies are being investigated. CD40-activated B cells (CD40-B) have been characterized previously as an interesting alternative because they present antigen efficiently and can be expanded by several logs from small amounts of peripheral blood. To determine the central technical challenges of cell-based vaccines we performed a single-patient analysis of 502 patients from DC-based tumour vaccine trials and identified at least three factors contributing to their limited efficiency: (1) lack of cell numbers; (2) lack of documented purity thus high contamination of bystander cells; and (3) lack of quality control and thus heterogeneous or unknown expression of important surface molecules such as major histocompatibility complex (MHC) and chemokine receptors. Based on these findings we re-evaluated the CD40-B approach in cancer patients. Here, we show that proliferation of B cells from cancer patients is equivalent to that observed in healthy donors. Purity is always > 90% after 2 weeks and remains stable for several weeks. They have comparable antigen-presenting capability determined phenotypically and by allogeneic mixed lymphocyte reaction. Expression of CCR7 and CD62L was detected in all samples and B cells migrated towards the relevant homing chemokines. Taken together, CD40-B cells from cancer patients can be expanded in virtually unlimited numbers at high purity and full function concerning antigen-presentation and migratory properties.

Early treatment failure is still a clinical challenge despite high cure rates in Hodgkin lymphoma (HL) patients. To identify the biological risk factors predicting early treatment failure, we performed a retrospective case-control study. Forty-seven pretherapeutic serum samples were available from 47 advanced stage HL patients with early treatment failure and from 47 matched controls in complete remission. All patients were treated within German Hodgkin Study Group phase 3 trials. Matching was done according to treatment, stage, age, gender, International Prognostic Score (IPS) and histological subtype. Pretreatment serum levels of 30 cytokines, chemokines and soluble receptors were determined using immunoassays and flow cytometer based cytometric bead arrays. Only interleukin-10 serum levels were significantly associated with early treatment failure after statistical correction for multitesting (paired-sign test, p = 0.0008). In summary, pretherapeutic interleukin-10 levels are associated with early treatment failure within 12 months after the end of treatment in advanced stage HL independently from known clinical factors such as age or IPS.

In solid tumors, leukemias, and lymphomas, increased frequencies of functional CD4+CD25high regulatory T cells (Treg cells) have been previously demonstrated. In healthy individuals, Treg cells consist not only of memory but also of naive T cells, which can undergo peripheral expansion and are characterized by a relative enrichment for autoreactive T-cell receptors. Here, we demonstrate in patients with premalignant monoclonal gammopathy of undetermined significance and patients with multiple myeloma that functional FoxP3+ Treg cells of naive, central, and effector memory phenotype as determined by CCR7 and CD45RA expression are significantly expanded. Low frequencies of T-cell receptor excision circles in naive Treg cells in both healthy controls and multiple myeloma patients point to peripheral expansion as the prominent mechanism of increased frequencies of naive Treg cells in these cancer patients. These findings strongly suggest that the increase of functional Treg cells in cancer patients is a response to the process of malignant transformation.

The CC thymus and activation-related chemokine (TARC) is a protein, which is highly expressed by Reed-Sternberg cells in Hodgkin's disease and is found in the majority of Hodgkin's disease patients. Within several trials conducted by the German Hodgkin study group, 62 Hodgkin's disease patients were elected based on availability of serum samples post and prior therapy to assess TARC levels by ELISA. TARC levels from 33 patients with continuous complete response (CCR), 20 patients with relapse, and nine patients with progressive disease (PD) were correlated with freedom from treatment failure and survival. As defined in healthy donors (mean value ± 2× SD), a TARC level of >500 pg/mL was considered as elevated. The median TARC levels of all patients at baseline and after completed primary treatment were 5,803 pg/mL (range, 116-73,074 pg/mL) and 663 pg/mL (50-24,709 pg/mL), respectively. TARC levels of patients with PD were higher than those of patients with CCR at baseline and after therapy. Baseline TARC correlated significantly with stage (P = 0.019), erythrocyte sedimentation rate (P = 0.004), leukocyte count (P < 0.001), and lymphocyte count (P = 0.026). A TARC level of >2,000 pg/mL after completed treatment was a significant risk factor for poorer survival (P = 0.02) but not for relapse. In conclusion, monitoring serum TARC levels in Hodgkin's disease patients may add valuable information about therapy success in Hodgkin's disease patients, especially those with PD and should therefore be prospectively evaluated in future trials.

Topotecan, a topoisomerase-I inhibitor is an active drug in the treatment of AML and MDS. To evaluate its toxicity and efficacy in a combination regimen with cytarabine, we conducted a clinical phase I/II trial in patients with relapsed acute myeloid leukemia (AML) or relapsed or newly diagnosed MDS RAEB, RAEB-t or CMML. Twenty-one patients (11 AML, 10 MDS/CMML) entered the study and were treated with 1.25 mg/m2 topotecan as continuous intravenous infusion daily for 5 days and cytarabine 1.0 g/m2 by infusion over 2 h daily for 5 days (TA). Cycles were repeated on day 28. The median observation time was 131 weeks (range: 36-196 weeks). A total of 37 cycles of TA were administered. In 1 patient, the dose of TA had to be reduced and in 1 patient, there was a treatment delay for the second cycle, both because of hematologic toxicity. The most frequent non-hematologic side-effect of TA was fever, which occurred in 17 patients (89%) with temperatures over 38 degrees C. None of the patients died due to any treatment-related toxicities, but 2 patients (10%) died within 1 month due to disease progression. A CR was achieved in 7 patients (33%), 3 of whom were MDS and 4 AML. A partial remission was reported in 8 patients (38%), no change of disease in 2 patients (10%) and progressive disease in 4 patients (19%). The median remission duration was 18 weeks (range 2-161 weeks) for MDS patients and 11 weeks (range 2-49 weeks) for AML patients. The time to progression for patients of 60 years and older (n = 10) was 16 weeks (range 2-49 weeks) and the survival was 32 weeks (range 2-119 weeks). TA is a feasible and efficacious chemotherapeutic combination for the treatment of MDS RAEB, RAEB-t, CMML and AML. For patients of 60 years and older, this regimen is also a safe option.

The detection of isolated tumor cells (ITC) in the bone marrow of patients with epithelial malignancies is an independant prognostic factor for several entities as breast cancer, colorectal cancer or non-small lung cancer. However, with conventional immunocytology using Ficoll density gradient and APAAP staining, only a small proportion of the bone marrow samples can be scanned for cytokeratin-positive (CK+) cells. To improve detection rates, we evaluated the enrichment of ITC by magnetic activated cell sorting (MACS) compared to regularly stained cytospins. Recovery experiments with a CK+ breast cancer cell line (SKBR3) were performed to calculate the MACS enrichment rate. Bone marrow was obtained by aspiration from 20 patients with carcinomas of epithelial origin and from 17 controls. ITC were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to cytokeratin 7 and 8. MACS of SKBR3 seeded in peripheral blood revealed average recovery rates of 62% and 48% and average enrichment factors of 104-fold and 8139-fold of the CK+ cells after one and after two separations, respectively. After immunomagnetic enrichment, CK+ cells were detected in 16 of 20 (80%) cancer patients, whereas only 7 (35%) patients showed CK+ cells without magnetic enrichment (P = 0.002). Ten of twelve (83%) patients with metastatic disease (stage M1) and six of eight (75%) patients without any overt metastases (M0) had CK+ cells in their bone marrow. None of the negative controls showed any CK+ cells. Enrichment with magnetically labeled anti cytokeratin antibodies increases the detection rate of epithelial cells in bone marrow of cancer patients compared to immunocytology.

Residual mediastinal masses are frequently observed in patients with Hodgkin disease (HD) after completed therapy, and the discrimination between active tumor tissue and fibrotic residues remains a clinical challenge. We studied the diagnostic value of metabolic imaging by 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) in detecting active mediastinal disease and predicting relapse. Twenty-eight HD patients with a residual mediastinal mass of at least 2 cm after initial therapy or after salvage chemotherapy were prospectively assigned to 29 examinations with FDG PET and were evaluated as 29 “subjects.” Patients were monitored for at least 1 year after examination and observed for signs of relapse. Median follow-up was 28 months (range, 16 to 68 months). A PET-negative mediastinal tumor was observed in 19 subjects, of whom 16 stayed in remission and 3 relapsed. Progression or relapse occurred in 6 of 10 subjects with a positive PET, whereas 4 subjects remained in remission. The negative predictive value (negative PET result and remission) at 1 year was 95%, and the positive predictive value (positive PET result and relapse) was 60%. The disease-free survival for PET-negative and PET-positive patients at 1 year was 95% and 40%, respectively. The difference was statistically significant. A negative FDG PET indicates that an HD patient with a residual mediastinal mass is unlikely to relapse before 1 year, if ever. On the other hand, a positive PET result indicates a significantly higher risk of relapse and demands further diagnostic procedures and a closer follow-up.

An accurate initial staging of patients with Hodgkin's disease (HD) is important for the evaluation of clinical stage and risk factors, which are crucial for the choice of an appropriate treatment. 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) is useful for detecting active tumor tissue in patients with lymphoproliferative diseases and may contribute to conventional staging methods in patients with HD. Twenty-two patients who presented with newly diagnosed HD underwent conventional staging methods including computed tomography (CT) as well as FDG PET. Lesions apparent in FDG PET and CT were correlated to each other. Seventy-seven lesions were observed either in PET or CT or in both. In 48 (62%) lesions PET and CT were both positive. In 20 (26%) sites, PET was positive and CT negative. Of 22 patients (18%) 4 were upstaged due to these positive PET findings, and as a result one patient received a different therapeutic regimen. PET failed to detect nine (12%) CT-positive sites in six patients. Statistically, these data are reflected by a sensitivity for PET and CT of 88% and 74%, respectively. Specificity of both imaging modalities was 100%. PET can contribute valuable information as an additional staging examination and led to an upstaging in some patients with primary HD. However, PET should not be used as the only imaging modality as it failed to detect CT-positive, active tumor regions in some cases.

Regulatory T cells (T(reg) cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of T(reg) cells was established. In IL-2 treated cancer patients a further T(reg)-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional T(reg) cells of a naïve phenotype--as determined by CCR7 and CD45RA expression--are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve T(reg)-cell pool. Higher frequencies of T-cell receptor excision circles in naïve T(reg) cells indicate IL-2 dependent thymic generation of naïve T(reg) cells as a mechanism leading to increased frequencies of T(reg) cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine T(reg) cells after IL-2 administration. These results point to a more complex regulation of T(reg) cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve T(reg) cells.

Abstract 4730 Background The DNA-based immunomodulator MGN1703 stimulates the innate and cellular immune system mainly via the TLR9-receptor. The results of the recent in vivo experiments showed potent anti-tumor efficacy of MGN1703 in several mouse tumor models in prophylactic and therapeutic settings as well as a good safety profile in various animals. Two investigator-initiated pilot trials of MGN1703 as adjuvant in patients with metastatic solid tumors also showed good safety and tolerability of the drug as well as a positive effect on the response rate in patients treated with MGN1703. Patients/Methods In this Phase 1 clinical trial MGN1703 is administered subcutaneously in escalating doses (0.25 mg, 2 mg, 10 mg, 30 mg, and 60 mg; 3-6 patients per group) either in a single or in a multiple (2x / week over 6 weeks) dose regimen. Patients with metastatic tumors of the following entities are recruited for the study, if no other standard treatment options are available: Colorectal cancer, breast cancer, lung cancer, renal cell carcinoma and melanoma. Primary endpoints are evaluation of the safety and tolerability of escalating single doses and of escalating multiple doses of s.c. administered MGN1703, determination of the maximum tolerated dose (MTD) and dose limiting toxicity (DLT), and recommendation of a dose for a Phase 2 trial in patients. Results Currently, 12 patients have been treated and evaluated in the single dose groups of 0.25 mg, 2 mg, 10 mg and 30 mg (3 patients each). In the multiple dose group, 4 patients have been treated with 0.25 mg, 3 patients with 2 mg, 3 patients with 10 mg and 3 patients with 30 mg MGN1703, so far. Therapy was well tolerated except for sporadic transient symptoms as mild redness and induration of injection sites in two patients, increase of temperature to 38 °C in one patient, and fatigue in two patients. In the 0.25 mg group, one patient showed a stable disease (SD, according to RECIST) after 6 weeks of treatment, and in the 2 mg group, 3 of 3 patients showed a SD after 6 weeks. Treatment results of the last 2 dosing groups are pending. The four patients, who responded to the treatment with MGN1703, were treated with MGN1703 for further 6 weeks within an extension phase of this clinical trial. Two of them still had a SD after 12 weeks of treatment. Conclusions MGN1703 showed safety and tolerability at dosages up to 30 mg so far. The detailed evaluation of clinical and immunological responses is still ongoing. There has been no DLT at this point of the Phase 1 trial. Disclosures: Weihrauch: MOLOGEN AG: Membership on an entity's Board of Directors or advisory committees. Schmidt:MOLOGEN AG: Employment. Tschaika:MOLOGEN AG: Employment. Wittig:MOLOGEN AG: Membership on an entity's Board of Directors or advisory committees.

4133 Background: The combination of chemotherapy and the anti-VEGF antibody bevacizumab (BV) is the current standard treatment for advanced colorectal cancer (CRC). Previous efforts to identify bio...

To evaluate, whether humoral hepatitis-B-vaccine non-responders also fail to mount a T cell response and to compare these results to normal vaccinees.Fourty-seven health care employees were enrolled in this study including all available non-responders (n = 13) with an anti-HBsAg titer10 kU/L and all available low-responders (n = 12) with an anti-HBsAg titer100 kU/L. Also, 12 consecutive anti-HBsAg negative pre-vaccination subjects were enrolled as well as 10 subjects (+7 from the vaccinated group) with titers1000 kU/L as controls. PBMC from all subjects were analyzed by IFN-gamma and IL-4 ELISPOT assays for the presence of hepatitis B surface antigen (HBsAg) reactive T cells.Non-responders and low-responders had no or only very limited T cell responses, respectively. Individuals responding to vaccination with the induction of a high anti-HBsAg titer showed a strong T cell response after the third vaccination. Surprisingly, these individuals showed response even before the first vaccination. T cell response to control antigens and mitogens was similar in all groups.Our data suggest that there is no general immune deficiency in non-/low-responders. Thus, we hypothesize that the induction of anti-HBsAg responses by vaccination is significantly dependent on the pre-existing T cell repertoire against the specific antigen rather than the presence of a general T cell defect.

To the Editor: Extramedullary plasmacytomas occur either as solitary (primary) tumors or as secondary manifestations of multiple myeloma. Involvement of the orbit is very rare in both entities.1 Pa...