Luitpold Distel

Survivin as a Radioresistance Factor, and Prognostic and Therapeutic Target for Radiotherapy in Rectal Cancer

Apoptosis levels have been shown to predict tumor response to preoperative radiochemotherapy in rectal cancer. Recently, the prominent role of survivin, a structurally unique member of the inhibitor of apoptosis protein family, has been shown in colorectal cancer tumorigenesis and prognosis. In this study, we investigated whether survivin plays a direct role in mediating radiation resistance. We used short interfering RNA molecules to decrease survivin in radioresistant SW480 and intermediately radioresistant HCT-15 colorectal cancer cells. This resulted in a significant decrease of survivin mRNA and protein expression with a maximum at 24 to 48 hours after transfection. If irradiated during this sensitive period, an increased percentage of apoptotic cells and an increased caspase 3/7 activity in parallel with a decreased cell viability and a reduced clonogenic survival was shown. These effects were more pronounced in the radioresistant SW480 cell line with a radiation-induced cytotoxicity enhancement factor at 10% and 50% survival of 1.8 to 2.2 for SW480 and 1.5 to 1.7 for HCT-15, respectively. Furthermore, transfection with survivin short interfering RNA increased levels of G2-M arrest and levels of DNA double-strand breaks in irradiated cells. These observations indicate that cell cycle and DNA repair mechanisms may be associated with apoptosis induction in tumor cells that are otherwise resistant to killing by radiation. In a translational study of 59 patients with rectal cancer treated with a combination of radiotherapy and chemotherapy, increased survivin expression was inversely related to the levels of apoptosis, and was also associated with a significantly higher risk of a local tumor recurrence.

Tumor-Infiltrating Cytotoxic T Cells but not Regulatory T Cells Predict Outcome in Anal Squamous Cell Carcinoma

Purpose: Tumor-infiltrating lymphocytes (TIL) are a possible prognostic factor in solid tumors. Cytotoxic TILs are generally considered as prognostically favorable, whereas regulatory T cells (Treg) may have adverse effects by virtue of their ability to inhibit effector cells. We have evaluated the effect of T-cell subsets on survival in patients with anal squamous cell carcinoma following radiochemotherapy. Methods: Biopsy specimens from 38 patients with anal carcinomas were evaluated using tissue microarrays and immunohistochemistry for the presence of tumor-infiltrating immune cells using CD3, CD4, CD8, and CD68 antibodies. Treg were identified using an antibody directed against the transcription factor FoxP3, and granzyme B served as a marker for cytotoxic cells. Intratumoral immune cells were enumerated using a semiautomatic image analysis program. Prognostic effect of TIL subsets was evaluated by the log-rank test comparing no evidence of disease survival for groups with high and low numbers using median values as cutoff. Results: CD3+ and CD4+ TILs influenced no evidence of disease survival: 3-year rates for patients with low numbers were 89% and 95%, respectively, and 54% (P = 0.02) and 48%, (P = 0.01), respectively, in cases with high numbers. Large numbers of tumor-infiltrating granzyme B+ cytotoxic cells had a significant negative prognostic effect (P = 0.008), whereas no effect was observed for Treg. Conclusions: TILs were identified as negative prognostic indicators in anal squamous cell carcinomas with granzyme B+ cytotoxic cells showing highest effect on outcome. This is possibly explained by the selection of therapy-resistant tumor cell clones. No prognostic influence of Treg was found. Knowledge of local immune responses is important for the development of immunotherapeutic strategies.

Prognostic impact of tumour-infiltrating Th2 and regulatory T cells in classical Hodgkin lymphoma†

Classical Hodgkin Lymphoma (cHL) is morphologically characterized by a small number of tumour cells, Hodgkin and Reed–Sternberg (HRS) cells, surrounded by numerous tumour‐infiltrating lymphocytes (TIL). The functional role of these TIL is still controversial. While generally considered to represent an anti‐tumour immune response, TIL in cHL might result from the profoundly deregulated immunity of cHL patients. Eighty‐seven cases of cHL were available to evaluate the prognostical significance of tumour‐infiltrating cytotoxic T lymphocytes (CTL), T helper 1 (Th1) cells, T helper 2 (Th2) cells and regulatory T cells (Treg). We confirm that in cHL the microenvironment is dominated by Th2 cells and Treg and show that large numbers of Th2 cells are associated with significantly improved disease‐free survival (p = 0.021) and event‐free survival (p = 0.012). Furthermore, a high ratio of Treg over Th2 cells resulted in a significantly shortened disease‐free survival (p = 0.025). These observations suggest that Treg may exert inhibitory effects on anti‐tumour immune responses mediated through Th2 cells and that Th2 cells may be more important for effective anti‐tumour immunity than anticipated. Copyright

CD24 Promotes Invasion of Glioma Cells In Vivo

Based on the hypothesis that adhesion molecules expressed on the surface of glioma cells mediate brain invasion, we examined the effect of CD24 on growth and migration of gliomas in vitro and in vivo. CD24, a glycosylphosphatidylinositol anchored, highly glycosylated adhesion molecule, is expressed in hematopoietic and neural cells. We found immunohistochemical expression of CD24 in human glioblastomas. We then established a clone from C6 rat glioblastoma cells, where mouse CD24 (also called heat stable antigen) is under control of a tetracycline-responsive promoter. In the presence of tetracycline (1 microg/ml) CD24 was downregulated by 20-fold. In vitro migration assays were performed on a basement membrane preparation (matrigel) and on myelin, the main substrates of in vivo glioma migration. While the cells were more motile on matrigel as compared with myelin, no relation between CD24 expression and motility was observed. We then transplanted the C6 clone into the striatum of nude mice and regulated CD24 expression via tetracycline in the drinking water (1 mg/ml). After 3 weeks, CD24 positive tumors of mice getting no tetracycline showed diffuse invasion of tumor cells in a brain area 10-fold larger than in CD24-suppressed tumors of mice receiving tetracycline. These data show that CD24 stimulates migration of gliomas in vivo and they suggest a role for this adhesion molecule in diffuse brain invasion of human gliomas.

Stromal regulatory T-cells are associated with a favourable prognosis in gastric cancer of the cardia.

BackgroundRecent evidence suggests that CD4+CD25+FoxP3+ regulatory T-cells (Treg) may be responsible for the failure of host anti-tumour immunity by suppressing cytotoxic T- cells. We assessed the prognostic significance of tumour infiltrating lymphocytes (TIL) in intestinal-type gastric cardiac cancer.MethodsTumour infiltrating lymphocyte (TIL) subsets and tumour infiltrating macrophages (TIM) were investigated in 52 cases using tissue microarrays. The interrelationship between the cell populations (CD3+, CD8+, CD20+, CD68+, GranzymeB+, FoxP3+) in different compartments and NED-survival was investigated (median follow-up time: 61 months).ResultsIntraepithelial infiltration with TIL and TIM including Treg was generally low and not related to NED-survival. However, patients with large numbers of FoxP3+ Treg in the tumour stroma (>125.9 FoxP3+TILs/mm2) had a median survival time of 58 months while those with low FoxP3+ TIL counts (<125.9 FoxP3+TILs/mm2) had a median survival time of 32 months (p = 0.006). Patients with high versus low stromal CD68+/FoxP3+ cell ratios in primary tumour displayed median survivals of 32 and 55 months, respectively (p = 0.008).ConclusionOur results suggest that inflammatory processes within the tumour stroma of gastric intestinal-type adenocarcinomas located at the gastric cardia may affect outcome in two ways. Tumour-infiltrating macrophages are likely to promote carcinogenesis while large numbers of Treg are associated with improved outcome probably by inhibiting local inflammatory processes promoting carcinogenesis. Thus, inhibition of Treg may not be a feasible treatment option in gastric adenocarcinoma.

Distribution of immune cells in head and neck cancer: CD8+ T-cells and CD20+B-cells in metastatic lymph nodes are associated with favourable outcome in patients with oro- and hypopharyngeal carcinoma

BackgroundTumour infiltrating lymphocytes (TIL) are generally considered to represent a host immune response directed against tumour antigens. TIL are also increasingly recognised as possible prognostic parameters. However, the effects observed are variable indicating that results cannot be extrapolated from type of tumour to another. Moreover, it has been suggested that primary solid tumours may be ignored by the immune system and that a meaningful immune response is only mounted in regional lymph nodes.MethodsWe have examined the local distribution of immune cells in tumour-related compartments in head and neck squamous cell carcinomas (HNSCC). In a second step, the prognostic impact of these cells on disease-free survival (DFS) was analysed. A total of 198 tissue cores from 33 patients were evaluated using tissue mircroarray technique and immunohistochemistry. Tumour-infiltrating immune cells were identified using antibodies specific for CD3, CD8, GranzymeB, FoxP3, CD20 and CD68 and quantified using an image analysis system.ResultsWe demonstrate a relative expansion of FoxP3+ regulatory T-cells (Treg) and of cytotoxic T-cells among tumour infitrating T-cells. We also show that intratumoural CD20+ B-cells are significantly more frequent in metastatic deposits than in primary tumours. Furthermore, we observed a reduced number of peritumoural CD8+ T-cells in metastatic lymph nodes as compared to univolved regional nodes suggesting a local down-modulation of cellular immunity. All other immune cells did not show significant alterations in distribution. We did not observe an association of tumour infiltrating immune cells at the primary site with outcome. However, increased numbers of intraepithelial CD8+ TIL in metastatic tumours as well as large numbers of peritumoural B-cells in lymph node metastases were associated with favourable outcome. Unexpectedly, no effect on patient outcome was observed for Treg in any compartment.ConclusionOur results suggest that alterations in lymphocyte distribution in regional lymph nodes rather than at the primary tumour site may be relevant for patient prognosis. Moreover, we demonstrate that in addition to cellular immunity humoral immune responses may be clinically relevant in anti-tumour immunity.

NODAL CT DENSITY AND TOTAL TUMOR VOLUME AS PROGNOSTIC FACTORS AFTER RADIATION THERAPY OF STAGE III/IV HEAD AND NECK CANCER

PURPOSE To determine whether the immunohistochemical expression of proliferation-associated antigens (proliferating cell nuclear antigen, MIB1) and the nuclear p53 reactivity in addition to total tumor volume, nodal CT density and T and N category are predictive for overall survival and locoregional tumor control in patients with squamous cell carcinoma of the head and neck region. MATERIALS AND METHODS Between October 1989 and September 1993, 87 patients with biopsy proven head and neck cancer were randomly allocated to receive radiation alone or simultaneous radiation and chemotherapy as part of a multicenter trial with a total of 298 randomized patients. There were only inoperable lesions in UICC (1992) stage III (8%) and IV (92%). Radiotherapy was delivered with 180 cGy twice daily up to a total dose of 7020 cGy in 51 days. Three cycles of 2340 cGy each were separated by a rest period of 11 days. Chemotherapy consisted of cis-DDP, 5-fluorouracil and leucovorin and was repeated on days 22 and 44. Routinely-processed paraffin-embedded sections were stained using monoclonal antibodies for detection of proliferation-associated antigens (MIB1 and PCNA) and p53 oncoprotein to determine the labeling index (LI). In addition, the total tumor volume and the percentage of necrosis were measured using CT data. The median follow-up was 3.9 years (range 1.9-5.0 years). RESULTS The overall survival and locoregional control for all 87 patients were 34 and 39% at 3 years, respectively. The addition of chemotherapy resulted in a better overall survival (27 versus 47%, P = 0.03) but did not influence locoregional control (31 versus 47%, P = 0.08). In univariate analysis, nodal CT density (P < 0.0001), total tumor volume (P < 0.0001), age (P = 0.001) and the MIB1-LI (P = 0.04) had a significant impact on overall survival. However, in the final Cox model only the nodal CT density (P = 0.0003) and age (P = 0.05) were independent prognostic factors for survival and only the nodal CT density (P = 0.0006) was an independent prognostic factor for locoregional control. The expression of the p53 oncoprotein was not found to have a clear predictive value. CONCLUSION Nodal CT density, total tumor volume and age will remain the relevant prognostic factors in stage III/IV head and neck cancer.

Normal V(D)J recombination in cells from patients with Nijmegen breakage syndrome.

The majority of antigen receptor diversity in mammals is generated by V(D)J recombination. During this process DNA double strand breaks are introduced at recombination signals by lymphoid specific RAG1/2 proteins generating blunt ended signal ends and hairpinned coding ends. Rejoining of all DNA ends requires ubiquitously expressed DNA repair proteins, such as Ku70/86 and DNA ligase IV/XRCC4. In addition, the formation of coding joints depends on the function of the scid gene encoding the catalytic subunit of DNA-dependent protein kinase, DNA-PK(CS), that is somehow required for processing of coding end hairpins. Recently, it was shown that purified RAG1/2 proteins can cleave DNA hairpins in vitro, but the same activity was also described for a protein complex of the DNA repair proteins Nbs1/Mre11/Rad50. This leaves the possibility that either protein complex might be involved in coding end processing in V(D)J recombination. We have therefore analyzed V(D)J recombination in cells from patients with Nijmegen breakage syndrome, carrying a mutation in the nbs1 gene. We find that V(D)J recombination frequencies and the quality of signal and coding joining are comparable to wild-type controls, as analyzed by a cellular V(D)J recombination assay. In addition, we did not detect significant differences in CDR3 sequences of endogenous Ig lambdaL and kappaL chain gene loci cloned from peripheral blood lymphocytes of an NBS patient and of healthy individuals. These findings suggest that the Nbs1/Mre11/Rad50 complex is not involved in coding end processing of V(D)J recombination.

Superparamagnetic iron oxide nanoparticles as radiosensitizer via enhanced reactive oxygen species formation

Internalization of citrate-coated and uncoated superparamagnetic iron oxide nanoparticles by human breast cancer (MCF-7) cells was verified by transmission electron microscopy imaging. Cytotoxicity studies employing metabolic and trypan blue assays manifested their excellent biocompatibility. The production of reactive oxygen species in iron oxide nanoparticle loaded MCF-7 cells was explained to originate from both, the release of iron ions and their catalytically active surfaces. Both initiate the Fenton and Haber-Weiss reaction. Additional oxidative stress caused by X-ray irradiation of MCF-7 cells was attributed to the increase of catalytically active iron oxide nanoparticle surfaces.

Squamous cell carcinoma of the oropharynx: Ki-67 and p53 can identify patients at high risk for local recurrence after surgery and postoperative radiotherapy

PURPOSE To assess the prognostic value of biologic (p53, Ki-67) and clinical factors in squamous cell carcinoma of the oropharynx after radical surgery and postoperative radiotherapy (RT). METHODS AND MATERIALS Between 1985 and 1995, a total of 102 patients with 104 tumor sites were entered onto the study. Fifty-five primary tumors (53%) involved the tonsils, 26 (25%) the soft palate, and 23 (22%) the base of the tongue. Median age was 53 years (range 36-80 years). The clinical T- and N-categories (UICC 1997) were: T1 (30), T2 (47), T3 (22), T4 (5), N0 (33), N1 (28), N2 (42), and N3 (1). Histologically-clear margins were achieved in all patients by initial surgery. Postoperative RT to the primary and regional lymphatics was given, to a total of 60 Gy in 6 weeks, and single daily fractions of 2 Gy. The expression of the nuclear p53- and Ki-67-labeling index (LI) was investigated by immunostaining using the monoclonal antibodies DO-7 and MIB 1. The nuclear p53-intensity (p53-I) was graded into 4 categories (0/+/++/) by densitometry. Median follow-up was 43 months (range 14-132 months). RESULTS Cancer-specific survival, disease-free survival, and locoregional tumor control rates were 74%, 69%, and 75%, respectively, at 5 years. Significant prognostic factors for disease-free survival were: T-category (T1/2: 77% vs. T3/4: 53%, p = 0.02), tumor site (tonsils: 79% vs. soft palate: 70% vs. base of tongue: 45%, p = 0.05), duration of RT (< or = 46 days: 80% vs. > 46 days: 60%, p = 0.04), Ki-67 LI (< or = 20%: 84% vs. > 20%: 49%, p = 0.006) and p53-I (0/+: 56% vs. ++/ : 79%, p = 0.008). A significant prognostic impact on locoregional control was noted for the duration of RT (< or = 46 days: 86% vs. > 46 days: 68%, p = 0.01), tumor site (tonsils: 88% vs. soft palate: 67% vs. base of tongue: 51%, p = 0.02), Ki-67 LI (< or = 20% LI: 87% vs. > 20% LI: 56%, p = 0.018), and the p53-I (0/+: 58% vs. ++/ : 88%, p = 0.0006). On multivariate analysis, the p53 nuclear intensity (p = 0.002) and the Ki-67 index (p = 0.01) remained the only significant factors for locoregional control. CONCLUSION Ki-67 labeling index above 20% and a weak p53 nuclear intensity (0/+) are both able to identify patients with squamous cell carcinoma of the oropharynx being at high risk for local recurrence after surgery and postoperative RT. Consequently, in this subgroup an intensification of treatment may be contemplated in prospective trials.