Carsten Zwick

Expression of SSX genes in human tumors

The HOM‐MEL‐40 antigen which is encoded by the SSX‐2 gene was originally detected as a tumor antigen recognized by autologous IgG antibodies in a melanoma patient. Expression analysis demonstrated that SSX‐2 is a member of the recently described cancer/testis antigen (CTA) class as it is expressed in a variety of different human neoplasms, but not in normal tissues with the exception of testis and a weak expression in the thyroid. Further studies demonstrated that SSX‐2 belongs to a gene family consisting of at least 5 homologous genes. We now report the analysis of the expression of all 5 SSX genes in 325 specimens of human neoplasms from various histological origins, using reverse transcription polymerase chain reaction (RT‐PCR). SSX‐1, ‐2, and ‐4 were found to be expressed in 8%, 15% and 15%, of the tumors, respectively, while the expression of the SSX‐5 gene was rare (7/325), and SSX‐3 expression was not detected. For defined tumor types, expression of at least one of the SSX family members was most frequently observed in head and neck cancer (75%), followed by ovarian cancer (50%), malignant melanoma (43%), lymphoma (36%), colorectal cancer (27%) and breast cancer (23%), while leukemias and the few cases of leiomyosarcomas, seminomas and thyroid cancers were found not to express any SSX gene. Int. J. Cancer 77:19–23, 1998.© 1998 Wiley‐Liss, Inc.

The role of sex and weight on rituximab clearance and serum elimination half-life in elderly patients with DLBCL

Pharmacokinetics of 8 doses of rituximab (375 mg/m(2)) given in combination with 2-week cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone/prednisolone (CHOP-14) was determined by ELISA in 20 elderly patients with diffuse large B-cell lymphoma (DLBCL) 10 minutes before and after each infusion and 1 week and 1, 2, 3, 6, and 9 months after the last infusion. Population pharmacokinetic modeling was performed with nonlinear mixed-effect modeling software (NONMEM VI). Concentration-time data were fitted into an open 2-compartment model and total clearance, central compartment volume, intercompartment clearance, and volume of distribution at steady-state (Vd(ss)) were investigated. Total clearance was 9.43 mL/h and Vd(ss) was 9.61 l. Rituximab clearance was reduced (8.21 mL/h vs 12.68 mL/h; P = .003) and elimination half-life was prolonged in women compared with men (t(1/2β) = 30.7 vs 24.7 days; P = .003). Body weight also affected Vd(ss) (0.1 l increase of Vd(ss) per kilogram above median of 75 kg). A sex-dependent effect and the higher weight of males contribute to their faster rituximab clearance, which might explain why elderly males benefit less from the addition of rituximab to CHOP than females. This trial was registered on www.clinicaltrials.gov as numbers NCT00052936, EU-20243 (RICOVER-60 Trial), EU-20534, and NCT00726700 (Pegfilgrastim Trial).

Analysis of the B-cell repertoire against antigens expressed by human neoplasms.

Summary: The screening of tumor‐derived expression libraries for antigens detected by high‐titer immunoglobulin G antibodies from the sera of patients with cancer by SEREX (serological identification of antigens by recombinant expression cloning) allows a systematic search for antigens of human cancers. SEREX has led to the identification of a multitude of new tumor antigens in many different tumor entities. According to their specificities, the antigens can be grouped into different classes, of which the cancer testis antigens appear to be the most attractive candidates for vaccine development. Serologically defined human tumor antigens facilitate the identification of antigenic peptides recognized by tumor‐specific T lymphocytes, thus providing a molecular basis for polyvalent peptide‐based and gene‐therapeutic vaccine strategies in a wide variety of human neoplasms. Moreover, many of the SEREX‐identified antigens seem to play a functional role in the pathogenesis of malignant disease. With more than 2000 antigens listed in the SEREX database, it appears that tumor antigens that have resisted discovery to date are expressed in only a small minority of tumors, thus limiting their clinical usefulness. Novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of non‐Hodgkins lymphoma patients.

Role of Radiotherapy to Bulky Disease in Elderly Patients With Aggressive B-Cell Lymphoma

PURPOSE R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) is standard care for aggressive B-cell lymphoma. A prospective trial was conducted to investigate the role of additive radiotherapy (RT) to bulky and extralymphatic disease. PATIENTS AND METHODS The best arm of the RICOVER-60 trial (6×R-CHOP-14+2R [R-CHOP administered once every 2 weeks plus two additional applications of rituximab] plus involved-field RT [36 Gy] to sites of initial bulky [≥ 7.5 cm] disease and extralymphatic involvement) was compared with a cohort receiving the same immunochemotherapy but without RT in an amendment to the RICOVER-60 trial (RICOVER-noRTh) in a prospective fashion. RESULTS After a median observation time of 39 months, 164 of 166 RICOVER-noRTh patients were evaluable. In a multivariable analysis of the intention-to-treat population adjusting for International Prognostic Index risk factors and age (> 70 years), event-free survival (EFS) of patients with bulky disease was inferior without additive RT (hazard ratio [HR], 2.1; 95% CI, 1.3 to 3.5; P = .005), with trends for inferior progression-free (PFS; HR, 1.8; 95% CI, 1.0 to 3.3; P = .058) and overall survival (OS; HR, 1.6; 95% CI, 0.9 to 3.1; P = .127). In a per-protocol analysis with 11 patients in RICOVER-noRTh excluded for receiving unplanned RT, multivariable analysis revealed HRs of 2.7 (95% CI, 1.3 to 5.9; P = .011) for EFS, 4.4 (95% CI, 1.8 to 10.6; P = .001) for PFS, and 4.3 (95% CI, 1.7 to 11.1; P = .002) for OS for patients not receiving RT to bulky disease. CONCLUSION Additive RT to bulky sites abrogates bulky disease as a risk factor and improves outcome of elderly patients with aggressive B-cell lymphoma. Whether RT can be spared in patients with (metabolic) complete remission after immunochemotherapy must be addressed in appropriately designed prospective trials.

Suboptimal dosing of rituximab in male and female patients with DLBCL

To determine the effect of gender on outcome, the male hazard ratio for progression-free survival (HRPFS-male) was determined in patients with diffuse large B-cell lymphoma (DLBCL). In young patients (MapThera International Trial study), HRPFS-male was 1.3 (P = .092) without and 1.1 (P = .660) with rituximab. In elderly patients (RICOVER-60 study), HRPFS-male was 1.1 (P = .348) with CHOP but increased to 1.6 (P = .004) with R-CHOP. The similar improvements of outcome in young patients were associated with similar rituximab clearances in young males and females (9.89 vs 10.38 mL/h; P = .238), whereas the greater benefit for elderly females was associated with a slower rituximab clearance (8.47 vs 10.59 mL/h; P = .005) and hence higher serum levels and longer exposure times, attributable to an age-dependent (P = .004) decrease of rituximab clearance in females but not males. Compared with elderly females, all other subgroups had significantly faster rituximab clearances and hence appear to be suboptimally dosed when rituximab is given at 375 mg/m(2). Although early results of pharmacokinetic-based prospective trials designed to exploit the full therapeutic potential of rituximab suggest that increased doses and/or prolonged exposure times can improve the outcome of elderly males with DLBCL, further studies are warranted that address the optimization of rituximab dose and schedule in all subgroups of DLBCL patients.

A novel tumour associated leucine zipper protein targeting to sites of gene transcription and splicing

We describe here the definition and characterization of antigen CT-8/HOM-TES-85 encoded by a previously unknown gene and identified by serological expression screening using antibodies from a seminoma patient. Intriguingly, the leucine zipper region of CT-8/HOM-TES-85 shows an atypical amphipathy with clusters of hydrophobic residues that is exclusively shared by the N-myc proto-oncogene. CT-8/HOM-TES-85 gene is tightly silenced in normal tissues except for testis. However, it is frequently activated in human neoplasms of different types including lung cancer, ovarian cancer, melanoma and glioma. Endogenous as well as heterogeneously expressed CT-8/HOM-TES-85 targets predominantly to the nucleus forming a distinctive speckled pattern of nuclear dots arranged in macromolecular structures. By co-localization studies these speckles were identified as loci of transcriptional activity and splicing, suggesting that CT-8/HOM-TES-85 may be involved in these processes. The aberrant expression of CT-8/HOM-TES-85 in human neoplasms might therefore be involved in cancer associated alterations of transcriptional or post-transcriptional processes and thus may disclose new mechanisms involved in the manifestation of the cancer phenotype.

Impact of Rituximab and Radiotherapy on Outcome of Patients With Aggressive B-Cell Lymphoma and Skeletal Involvement

PURPOSE To study clinical presentation, outcome, and the role of radiotherapy in patients with aggressive B-cell lymphoma and skeletal involvement treated with and without rituximab. PATIENTS AND METHODS Outcome of patients with skeletal involvement was analyzed in a retrospective study of nine consecutive prospective trials of the German High-Grade Non-Hodgkin lymphoma Study Group. RESULTS Of 3,840 patients, 292 (7.6%) had skeletal involvement. In the MabThera International Trial (MInT) for young good-prognosis patients and the Rituximab With CHOP Over 60 Years (RICOVER-60) study for elderly patients, the randomized addition of rituximab improved event-free survival (EFS; hazard ratio for MInT [HRMInT] = 0.4, P > 001; hazard ratio for RICOVER-60 [HRRICOVER-60] = 0.6, P > .001) and overall survival (OS; HRMInT = 0.4, P < .001; HRRICOVER-60 = 0.7, P = .002) in patients without skeletal involvement, but failed to improve the outcome of patients with skeletal involvement (EFS: HRMInT = 1.4, P = .444; HRRICOVER-60 = 0.8, P = .449; OS: HRMInT = 0.6, P = .449; HRRICOVER-60 = 1.0, P = .935). Skeletal involvement was associated with a worse outcome after cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) plus rituximab (HREFS = 1.5, P = .048; HROS = 1.1; P = .828), but not after CHOP without rituximab (HREFS = 0.8, P = .181; HROS = 0.7, P = .083). In contrast to rituximab, additive radiotherapy to sites of skeletal involvement was associated with a decreased risk (HREFS = 0.3, P = .001; HROS = 0.5; P = .111). CONCLUSION Rituximab failed to improve the outcome of patients with diffuse large B-cell lymphoma with skeletal involvement, although our data suggest a beneficial effect of radiotherapy to sites of skeletal involvement. Whether radiotherapy to sites of skeletal involvement can be spared in cases with a negative positron emission tomography after immunochemotherapy should be addressed in appropriately designed prospective trials.

Optimization of Rituximab for the Treatment of Diffuse Large B-Cell Lymphoma (II): Extended Rituximab Exposure Time in the SMARTE-R-CHOP-14 Trial of the German High-Grade Non-Hodgkin Lymphoma Study Group

PURPOSE To study pharmacokinetics, toxicity, and efficacy of prolonged rituximab exposure in elderly patients with diffuse large B-cell lymphoma (DLBCL). PATIENTS AND METHODS In the SMARTE-R-CHOP-14 trial, rituximab 375 mg/m(2) was administered, together with six cycles of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone on a 14-day schedule (6×R-CHOP-14), on days -4, 0, 10, 29, 57, 99, 155, and 239. Pharmacokinetics and outcome were to be compared with those of patients who had received 6×R-CHOP-14 in combination with eight 2-week applications of rituximab in the RICOVER-60 (Rituximab With CHOP Over Age 60 Years) trial. RESULTS The complete response (CR)/unconfirmed CR rate was 85% in 189 evaluable patients, 90% for 90 good-prognosis patients (International Prognostic Index [IPI], 1 or 2), and 81% for 99 poor-prognosis patients (IPI, 3 to 5); 3-year event-free survival (EFS) was 71%, 75%, and 67%, respectively; and 3-year overall survival (OS) was 84%, 88%, and 80%, respectively, with no differences between men and women. The preplanned historical comparison with 306 RICOVER-60 patients (good prognosis, n = 183; poor prognosis, n = 123) revealed no outcome differences for all and good-prognosis patients; however, the longer exposure time in SMARTE-R-CHOP-14 compared with RICOVER-60 was associated with better 3-year EFS (67% v 54%) and OS (80% v 67%) in poor-prognosis patients. CONCLUSION Extended rituximab exposure compared with eight 2-week applications in combination with 6×R-CHOP-14 significantly improved outcome of elderly poor-prognosis patients without increasing toxicity. To our knowledge, results obtained with the SMARTE-R-CHOP-14 rituximab schedule are the best reported for elderly patients with DLBCL to date. In the subgroup of poor-prognosis patients treated with extended rituximab exposure, the outcome seemed superior to that of a similar historical cohort of patients treated with 6×R-CHOP-14 plus 2-week rituximab, with similar toxicity. A randomized comparison of the two schedules is warranted.

Randomized comparison of pegfilgrastim day 4 versus day 2 for the prevention of chemotherapy-induced leukocytopenia

BACKGROUND To study the effects of deferring pegfilgrastim until day 4 on the reduction of chemotherapy-induced leukocytopenia. PATIENTS AND METHODS Patients of age 61-80 years with aggressive lymphoma were randomly assigned to receive 6 mg pegfilgrastim on day 2 or 4 of a 2-week chemotherapy regimen (R-CHOP-14). RESULTS Two hundred and ninety-two and 313 chemotherapy cycles were evaluable in 103 patients. Post-nadir pegfilgrastim serum levels were higher after day 4 than after day 2 application. This was associated with an attenuated leukocyte nadir after day 4 pegfilgrastim and there were fewer days with leukocytes <2 × 10(3)/mm(3) compared with day 2 pegfilgrastim. Grade 3 and 4 leukocytopenias (70% versus 43.3%; P < 0.001) and grade 4-only leukocytopenias (47% versus 20.5%; P < 0.001) were more frequent after day 2 pegfilgrastim. There were more chemotherapy cycles with grade 3 and 4 infections after day 2 than day 4 pegfilgrastim (9.4% versus 6.0%; P = 0.118). Interventional antibiotics were given more often after day 2 than after day 4 pegfilgrastim (30.7% versus 21.9% of cycles; P = 0.008). There were five deaths during leukocytopenia after day 2 and none after day 4 pegfilgrastim (P = 0.027). CONCLUSIONS Administration of pegfilgrastim on day 4 was more effective in reducing severe leukocytopenias and resulted in fewer deaths during leukocytopenia. Pegfilgrastim should be given on day 4 to better exploit its myeloprotective potential.

Serological immune response to cancer testis antigens in patients with pancreatic cancer

Serological screening approaches have allowed for the identification of a large number of potentially relevant tumor antigens in cancer patients. Within this group, cancer testis antigens represent promising targets for cancer immunotherapy, since they are widely expressed in a variety of human cancer entities. In pancreatic cancer, however, there are only few data available about the expression pattern and serological response to cancer testis antigens and other serological‐defined tumor antigens. Therefore, we investigated the IgG antibody response against 11 cancer testis antigens (SCP‐1, GAGE, LAGE‐1a,‐1b, CT‐7, NY‐ESO‐1, SSX‐1‐5) recombinantly expressed on yeast surface (RAYS) in patients with pancreatic cancer (n = 96), chronic pancreatitis (n = 18) and healthy donors (n = 48). We found in 14% of all patients antibody responses to SCP‐1, but not to other cancer testis antigens (GAGE, LAGE‐1a,‐1b, CT‐7, NY‐ESO‐1, SSX‐1‐5). Antibody response correlated with the expression of SCP‐1 in the primary tumor of the respective patient as shown by RT‐PCR, immunohistochemistry and Western blot. In contrast, no serological response to cancer testis antigens was observed in healthy donors. The humoral immune response against SCP‐1 was associated with the size of tumor, but not with other clinico‐pathological parameters such as histology, stage, presence of lymph node metastases, grading, age, gender or gemcitabine treatment. In conclusion, antibody response to cancer testis antigen SCP‐1 is found in a proportion of pancreatic carcinoma patients. These results indicate that identification of additional tumor antigens by serological screening of tumor cDNA expression libraries by RAYS is a promising goal in pancreatic cancer.