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Manuscript Number: AOHE-D-10-00147 Title: Impressive response to temsirolimus in a patient with chemotherapy refractory diffuse large Bcell non-Hodgkin's lymphoma Article Type: Letter to the Editor

Introduction Enhanced progenitor proliferation, bone marrow (BM) hypervascularization and disturbed immune regulation contribute to the pathogenesis of myelodysplastic syndromes (MDS). Inhibition of mammalian-target of rapamycin (mTor) by temsirolimus (TEM) might be a promising strategy to target these disease-specific cellular alterations. We report on the effects of single agent TEM on the clinical course as well as on immune composition and BM vascularization of MDS patients treated within the prospective, multicenter “TEMDS”-trial (NCT01111448). Patients, Materials and Methods Twenty patients being either IPSS low/int-1 MDS (n = 9) or IPSS int-2/high after azacitidine failure were treated with TEM at a dose of 25 mg/week in the absence of toxicity or disease progression. BM was reevaluated after 4 months of treatment with the option of TEM continuation for a maximum of 12 months in responding patients. Translational research within this study included flowcytometry-based measurement of changes in T-cell composition as well as determination of cytokine levels and BM-vascularization prior to and after TEM. Results Of 20 patients treated, 15 discontinued TEM treatment prematurely due to intolerable side effects (n = 11), infectious complications (n = 3), or progression to AML (n = 1). Fatigue, stomatitis and profound leukopenia were the most frequent adverse events. A total of 13 severe adverse events were encountered in 10 patients and 1 patient died of infectious complications during TEM treatment. Of the 5 patients who were treated for at least 4 months and underwent regular BM reevaluation, none showed signs of response according to IWG criteria. TEM treatment resulted in a remarkable, although non-significant, decrease in total number of lymphocytes in the pB (pre: 74.6%, post: 48.4%, p = 0,083) and BM (pre: 23.5% post: 20.1%, p = 0.123). Within the T-helper cell compartment a trend towards an increase in regulatory T-cell (Treg) frequency was observed (pB: pre: 6.0 %, post: 6.4 %, p = 0.083). Moreover, the balance between naive (CD45RA+/CD45RO-) and activated/memory (CD45RA-/CD45RO+) Treg shifted significantly in favor of the latter (p = 0.004). Plasma analysis in BM and pB revealed, that these changes were obviously not mediated by alterations in TGFβ plasma levels. In a total of 12 assessable patients, a significant (p = 0.006) decrease of BM vascularization was observed after treatment with TEM for a median of 5 weeks (Fig. 1). There were, however, no changes in the medullary or peripheral blood VEGF concentration (data not shown). Conclusions Selective inhibition of the mTOR signaling cascade in MDS patients results in specific alterations of the composition of T-cell subsets as well as BM vascularization. Given the absence of any hematological response we suggest that these drug-induced modifications cannot alter the natural course of the disease. Disclosures: Wermke: Pfizer: Research Funding. Off Label Use: Temsirolimus is licensend for the treatment of MCL and RCC but not MDS. Platzbecker:Pfizer: Research Funding.

2007 Background: Age is the most important therapy-independent prognostic factor in patients with primary central nervous system lymphoma (PCNSL). Here we aimed at providing an analysis of the impact of higher age on response to therapy, toxicity, and survival in the largest PCNSL trial ever performed to date. Methods: Response to therapy, toxicity and survival of PCNSL patients enrolled in the G-PCNSL-SG-1 trial evaluating the role of radiotherapy after high-dose methotrexate (HD-MTX)-based chemotherapy were monitored. Subjects aged 70 or more were compared to younger patients. Results: Of all eligible patients (n=526), 126 (24%) were aged 70 or more. In the per protocol population, 66 of 318 patients (21%) were at least 70 years old. Among the eligible patients, the rate of complete and partial responses (CR+PR) to HD-MTX-based chemotherapy was 44% in the elderly compared to 57% in the younger patients (p=0.016). A higher rate of grade III/IV leukopenia was observed in the elderly (34% versus 21%, p=0.007). Also, death on therapy was more frequent (18% versus 11%; p=0.027) in these patients. In contrast, there was no other major age-dependent toxicity. Survival analyses revealed shorter progression-free survival (PFS) (4.0 versus 7.7 months, p=0.014) and overall survival (OS) (12.5 versus 26.2 months, p

Abstract 3125 Background: PCNSL is a rare form of extranodal diffuse large B-cell lymphoma (DLBCL). Relapses after initial therapy are usually within the CNS. Systemic relapses (SR) have been reported only occasionally. We conducted a systemic analysis of immunocompetent patients with PCNSL und SR using both clinical data and tumor samples at first diagnosis and SR to determine clonal relatedness. Methods: Records of patients included in a multicenter therapy trial (n=411) and all PCNSL patients treated at our institution outside the trial after 1993 (n=39) were reviewed for SR. PCR analysis of the rearranged Ig heavy-chain (IgH) and light-chain (IgL) genes was performed according to the BIOMED-2 protocol. Result: All patients had received high-dose methotrexate based chemotherapy with our without adjuvant whole brain irradiation as first line therapy. Of 450 patients 26 (5.8%) were identified with SR. Median time from PCNSL diagnosis to SR was 14.5 months (range 2–61). SR was extranodal in 22 (85%) patients: testicular n=3, gastric n=3, mamma n=3, liver n=2, bone/bone marrow n=3, intestinal n=1, [sub-]cutaneous/muscular n=5, pancreatic n=1, pulmonary n=1. Four patients had concomitant CNS relapse. Median overall survival from diagnosis was 38 months, from SR 9.8 months. Causes of death were SR in 13, CNS relapse in 3, toxicity of chemotherapy in 3 and unknown in 1 patients; 6 patients were alive at last follow up. All 26 patients initially had DLBCL. SR was biopsied in 20 patients: 19 had DLBCL, 1 a Burkitt-like DLBCL. DNA of 7 patients was sufficient for PCR analysis. The same clone was found in the CNS and SR in 4 patients, in 3 patients the clones were different. Clonally related SR occurred after 4, 8, 14 and 25 months; the unrelated SR after 30, 35 and 36 moths.PCR products in 2 of 4 clonally related samples were suitable for sequencing. One case exhibited 6 identical somatic mutations in both biopsies. The second case harbored 35 mutations in the CNS sample and 45 in the SR sample. Here 26 mutations were identical in both samples, 3 involved different nucleosides, 16 were only present in SR, and 6 mutations exclusively present in the CNS sample. Conclusion: SR is a rare and frequently fatal complication in PCNSL. The localization of SR is predominantly extranodal. A proportion of SR is not clonally related to the primary tumor and then most likely represent a secondary lymphoma. In clonally related SR the presence of somatic mutations unique to the CNS tumor and the SR might be explained by a common ancestor, whose localization however remains unknown. Disclosures: No relevant conflicts of interest to declare.

Background: The bifunctional protein survivin is a powerful prognostic marker in GEP-NET disease as measured by immunohistochemical methods in tumor tissues. The present study was focused to investigate if survivin can also be detected in the serum of GEP-NET patients and to evaluate its potential diagnostic role. Aim: To establish a method by which the serum levels of survivin can be used as a predictive value in the treatment outcome of patients with GEP-NET disease. Materials and Methods: In 2007, serum samples were obtained from patients with well-differentiated GEP-NET disease (n=22) either at Charité-Campus Benjamin Franklin (n=8) or at the University Hospital of Hamburg-Eppendorf (n=14), hematological malignancies (HM) and solid cancers (n=22), as well as healthy volunteers (n=25), the last all obtained at the Charité. All patients had given their informed consent to this pilot study. The patients of the GEP-NET group had either stable disease with metastasis, predominantly to the liver (n=14), or were progressive at the time of venipuncture (n=6). 9 foregut tumors, 10 midgut tumors and 3 hindgut tumors were included, a third of those functionally active. Most of the patients received any kind of bio- or chemotherapy. The mean age of GEP-NET patients was 61.1 years (range 29–81), whereas the mean age of patients with HM and solid tumors was 55.9 years (range 30–82). Enzyme-linked immunosorbent assay (ELISA) analysis was used for determination of survivin protein serum levels. Results: There was a statistically significant increase of the survivin protein serum levels in patients with HM and solid cancers compared to the healthy controls (p=0.009). The difference in survivin serum levels between healthy controls and patients with GEP-NETs did not reach statistical significance (p=0.089). No correlation to clinical features like localization of the tumor, functional activity, Chromogranin A levels or kind of therapy was found. However, survivin serum levels tended to be higher in patients with a greater tumor burden. Conclusion: This is the first study to quantify survivin protein levels in the serum of GEP-NET patients. The results of this small pilot study should be evaluated in further clinical trials by correlating survivin serum levels before and after therapy in order to define its potential diagnostic and prognostic use. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B44.

Introduction: The frequency of meningeal dissemination (MD) in primary central nervous system lymphoma (PCNSL) reported in the literature differs. The optimal management and the prognostic impact of MD have not been defined. Methods: Data on MD from all 92 immunocompetent patients primarily diagnosed with PCNSL at our institution between January 1994 and February 2007 were retrospectively analysed for MD evaluation. All patients received cranial MRI or CT, and in 71 patients CSF was obtained by lumbar puncture. High-dose methotrexate was the intended treatment in all patients. Results: Cerebrospinal fluid (CSF) was obtained by lumbar puncture in 71 patients (median age 62 years) of a total of 92 patients with PCNSL. Cytomorphologic CSF examination was performed in 63, immunophenotyping in 32 and PCR of the CDR3 region in 37 patients. Eight of 63 patients (13%) had positive cytomorphology, one of 32 (3%) positive immunocytology and 6 of 37 (16%) had monoclonal B-cells on PCR analysis. Evidence of MD on MRI or CT was found in three of the 71 patients (4%). Altogether, 11 (16%) patients had evidence of MD diagnosed by either of the methods used. Median CSF cell count was 5/μl (range, 0–237; n=66), and median protein concentration 76 mg/dl (range, 93–4117; n=55). An elevated cell count (>5/μl) was found in 27 (41%) patients, an elevated protein (>45 mg/dl) in 44 (80%). No significant correlation was found between proof of MD and CSF pleocytosis or elevated protein. Patients with MD did not differ from other patients with respect to age, gender, clinical presentation, performance status, CSF cell count and protein concentration, number of intracerebral lesions, lesion location or histology. After a median follow-up of 56 months, median EFS was 26 (95% CI, 4.3 to 47.7) months for MD patients and 34.1 (95% CI, 26.2 to 42) months for those without MD, p=0.26 (log-rank). Of 21 patients with relapse after initial treatment response, 16 had isolated cerebral relapse, one isolated meningeal relapse, two systemic relapse (one with cutaneous, and one with testicular manifestation), and one concomitant systemic (lung/thoracic wall) and cerebral relapse. Of the four MD patients with relapse after initial response, three had cerebral relapse, and one had systemic (cutaneous) relapse. The median overall survival (OAS) of patients with MD was 45.5 months (95% CI, 16.6 to 74.4), of those without MD 42.5 months (95% CI, 35.8 to 49.2) (p=0.27). Conclusions: The frequency of MD detected in our cohort was low despite the use of highly sensitive diagnostic methods. No impact on either EFS or OAS was seen for MD.

Background: The mechanisms leading to development of CNS lymphoma (CNSL) are still poorly understood. Homing of malignant lymphocytes to the CNS may play an important role in the pathogenesis of CNSL. Expression of chemokine receptors CXCR4 and CXCR5 has been shown on tumor cells of primary CNSL. In this study we examined the level of chemokines CXCL12 and CXCL13 which are ligands of CXCR4 and CXCR5 respectively in the CSF of CNSL patients and compared it to non-CNSL controls. Methods: Lumbar CSF and peripheral blood from 16 patients with primary and one with secondary CNSL, as well as control CSF from 8 patients with hydrocephalus, glioblastoma, peripheral lymphoma without CNS involvement or unspecific neurological symptoms were collected, and after centrifugation supernatant and serum were cryopreserved at −80°C until analysis. Chemokine concentrations were measured using commercially available ELISA kits (CXCL13/BCA-1 and CXCL12/SDF-1α Quantikine Immunoassay, R&D Systems, Minneapolis) according to the manufacturer’s instructions. Results were confirmed by duplicate measurements. Results: 22 CSF samples and 11 serum samples from CNSL patients and nine CSF samples from controls were evaluated (see table). There was no correlation between serum and CSF chemokine levels in CNSL patients. Median concentrations of both CXCL12 and CXCR13 were higher in patients with CNS lymphoma compared to controls, CXCL13 being significantly raised (p=0.0019). In one CNSL patient three serial CSF samples could be analysed and a ten-fold decrease of CXCL12 during succesfull chemotherapy was found whilst concentration of CXCL13 remained stable. Conclusion: CXCL12 and CXCL13 can be measured in the CSF of CNS lymphoma patients at higher concentration as compared to non-CNSL patients. Analysis of a larger population is warranted in order to define the role of these chemokines in CNSL and to establish their value for diagnosis and follow up. Serum CSF CNSL patients CNSL patients controls p range median (range) median (range) CXCL12 1788–2933 pg/ml 481 (55–2079) pg/ml 115 (0–2442) pg/ml 0.067 CXCL13 36–657 pg/ml 457 (18–657) pg/ml 16.9 (0.6–187) pg/ml 0.00019

Background This is a retrospective, long-term single-center analysis of immunocompetent patients with PCNSL treated at our institution. Methods All 72 consecutive patients diagnosed with PCNSL between January 1994 and February 2005 were scheduled to receive high-dose methotrexate (HDMTX; 1.5–4 g/m2) based chemotherapy. Results The median age of the patients was 62 years, the median Karnofsky performance status (KPS) was 70%. Twelve patients did not receive HDMTX based chemotherapy due to poor physical condition or renal insufficiency. Of 60 patients treated with HDMTX based chemotherapy, 9 were followed by whole brain irradiation (WBI). Of 54 patients evaluable for response 35 (65%) responded (52% CR and 13% PR), and 19 (35%) did not. At a median follow-up of 58.7 months, median progression-free survival (PFS) was 9 months, and median overall survival (OAS) was 41.4 months. Eight patients have survived 60 months or longer (median age 48, range 19–76; median KPS 80, range 40–100), 5 of whom relapsed after primary therapy at a median of 5 months (range, 3–39.7) after initial diagnosis. According to the Memorial Sloan-Kettering Cancer Center (MSKCC) prognosis score, patients could be divided into three groups with significantly different OAS: 52.9 months for patients younger than 50 years, 42.4 months for patients ≥50 years and with KPS ≥70, and 5.2 months for patients ≥50 years and KPS Conclusions This study providing long-term data shows that a relatively moderate HDMTX-based chemotherapy can result in prolonged survival and probably cure even in older and early relapsing patients. However, the probability of late neurotoxicity with prolonged survival is considerable. The comparison of our results to other retrospective studies underscore the importance of treating PCNSL patients preferably at experienced institutions. The MSKCC score proved useful to predict survival.

3046 Background: The trifunctional antibody catumaxomab specifically binds EpCAM+ tumor cells, CD3+ T lymphocytes and accessory cells via FcγR I/III. Thus the antibody induces tumor specific cell mediated cytotoxicity in vitro and in vivo. Following last years’ ASCO presentation on a phase I/II trial showing safety and efficacy of repetitive intrapleural administration of catumaxomab in patients with EpCAM positive, malignant pleural effusion (MPE), we now present data on the responsiveness of immune cells from pleural fluid to catumaxomab. Methods: Pleural fluid of patients with EpCAM-positive MPE treated with i.pl. catumaxomab was collected before treatment. Cells were harvested from the fluid and cultured ± 100 ng/ml catumaxomab using an in vitro proliferation assay. After 72 h of culture, proliferation of T cells (CD4+ and CD8+) and monocytes (CD11c+) was determined. In addition, cell supernatants after 24h incubation ± catumaxomab were analysed for their TH1/TH2 cytokine profile (IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF- a). Results: Incubation in presence of catumaxomab led to a pronounced increase of CD4+ CD8+ and CD11+ cell numbers indicating a proliferation of these cells, whereas cultures without catumaxomab showed no proliferation of immune cells. Analysis of supernatants after 24 h revealed levels of IL-2, IL-6, IFN-γ and TNF-a, from cells incubated with catumaxomab that were distinctly higher than in cultures without catumaxomab. Conclusions: The immunologic nature of catumaxomab-induced responses in patients with pleural effusion accompanied by a reduction of tumor cells, could be underlined impressively with in vitro data obtained from pleural cells showing catumaxomab-induced proliferation of T cells and accessory cells and TH1-directed cytokine secretion. The data are equivalent to results observed in the peritoneal fluid of ascites patients. [Table: see text]

12524 Background: CNS involvement is a grave complication of malignant lymphoma. Therapy traditionally includes intrathecal chemotherapy, radiotherapy and systemic chemotherapy for concomitant systemic lymphoma. However, optimal therapy has not been established thus far for CNS lymphoma. In this pilot study a combination of systemic high-dose (HD) methotrexate (MTX) and ifosfamide (IFO) was evaluated for toxicity and response rate. Methods: 10 patients (pts.) with CNS relapses of aggressive lymphoma (5 nodal DLBCL, 4 PCNSL/PIOL and 1 T-NHL) were treated. Nine pts. had intracerebral lesions, the meningeal compartment was involved in three. Three pts. had systemic manifestations. Median age was 63 years (32 - 83). Therapy consisted of 4g/m2 MTX (4h infusion on day 1) with dose adjustment for creatinine clearance (CC) < 100ml/min (CC/100 * 4g/m2) and 1.5 to 2g/m2/day IFO (3h infusion day 3 to 5). Supportive therapy included mesna and a leucovorin rescue beginning at 24h after start of MTX. Two pts. were treated with intrathecal chemotherapy before MTX/IFO. Results: A median of 4 cycles chemotherapy was administered. Treatment response was documented in 9 pts. (3 CR, 6 PR), and one patient had stable disease. The toxicity was mainly hematologic with grade 3/4 neutropenia in 6 pts., grade 3/4 thrombocytopenia in 4 pts. and grade 3 anemia in 2 pts. One pat. died from sepsis in neutropenia and one pat. with long-term corticosteroid therapy developed an aspergillus pneumonia grade 3. Nephrotoxicity grade 1/2 was observed in 6 pts.. Four pts. were further treated with HD chemotherapy followed by autologous stem cell transplantation or salvage radio-/chemotherapy. A median progression free survival of 6.5+ months (range 1–41+) for all pts. has been reached with persisting remission after 4 to 41 months in 5 pts.. Conclusions: Systemic combination therapy with MTX and IFO is feasible and exhibits a promising activity in CNS relapses of aggressive lymphoma. No significant financial relationships to disclose.

Background: Salvage treatment has not yet been established in primary CNS lymphoma (PCNSL). The introduction of anti-CD20 monoclonal antibody rituximab expanded treatment options in systemic B-cell lymphoma. Due to its large molecular size, rituximab poorly penetrates the blood-brain-barrier (BBB) limiting its use in PCNSL. According to FDG-PET investigations, however, the BBB is initially leaky in PCNSL and reconstitutes after therapy-induced tumor shrinkage. We treated patients with relapsed/resistant PCNSL with a single course of Y-90 anti-CD20 antibody ibritumomab tiuxetan and evaluated the penetration of the antibody into the tumor. Methods: Immunocompetent patients with histologically confirmed, therapy resistant or recurrent PCNSL after at least one pretreatment, and adequate cardiac and bone marrow function were treated with rituximab 250 mg/m2 on day -7 and day 0, followed by Y-90-ibritumomab tiuxetan 15 MBq/kg IV. In three patients single photon emission computed tomography (SPECT) target imaging with gamma-emitting 111-Indium-ibritumomab tiuxetan was performed repeatedly 4 - 168 hours after Y-90-ibritumomab tiuxetan. Response evaluation by contrast-enhanced magnetic resonance imaging (MRI) and fluorodeoxyglucose positron emission tomography (FDG-PET) was scheduled before, one month and two months after treatment as well as every three months thereafter in responders. Results: Seven patients with a median age of 46 years (range 40–63) and a median number of 4 (range 1–6) previous therapies received antibody treatment. SPECT indicated target accumulation in the tumor starting 48 hours and still ongoing 7 days after injection of 111-Indium-ibritumomab tiuxetan. Response was observed in four of seven patients: one uncertain complete response (uCR) lasting 18+ months, two CR (0.5 and one month), and one partial response (0.5 months). Relapse usually occurred distant of target lesions. Three patients had disease progression. Toxicities were leukopenia CTC grade 3/4 (n=6), pneumonia grade 3/4 (n=2) with one fatal outcome, and thrombocytopenia grade 3/4 (n=6). Conclusions: We provide first evidence of Y-90-ibritumomab tiuxetan penetration into PCNSL with target accumulation of the antibody in SPECT. However, responses were mostly of short duration in this heavily pretreated patient cohort with relapses occurring distant of target lesions. Hematotoxicity up to CTC grade 4 was the most common side-effect. Further investigation within a phase II study is warranted.

755 Background: The ubiquitin-proteasome pathway plays an essential role in intracellular degradation of key regulatory proteins that can affect cell cycle, angiogenesis, adhesion and apoptosis. Bortezomib (VELCADE, PS-341) is a first-in class selective inhibitor of the 26S proteasome. The oral fluoropyrimidine capecitabine (Xeloda) has shown substantial activity in taxane and/or anthracycline pretreated patients with metastatic breast cancer. This phase I trial was initiated to determine the maximum tolerated doses (MTD), efficacy and tolerability of combined treatment with capecitabine and bortezomib in patients with metastatic breast cancer previously treated with taxanes and/or anthracyclines. Methods: Patients were treated with bortezomib (1.0–1.3 mg/m2; days 1, 4, 8 & 11) and capecitabine (1500–2500 mg/m2, days 1–14) in 3-weeks intervals. Dose limiting toxicity (DLT) was defined as grade (G) 4 platelets, any G platelets with bleeding, G4 ANC >4 days, febrile neutropenia, any >G2 non-hematologic toxi...