Low-dose ipilimumab plus nivolumab combined with IL-2 and hyperthermia in cancer patients with advanced disease: exploratory findings of a case series of 131 stage IV cancers - a retrospective study of a single institution.

The 3-year overall survival (OS) rate of patients with previously treated or untreated stage III or IV melanoma has by now reached 63% using ipilimumab and nivolumab therapy. However, immune-related adverse events (irAEs) of grade 3 or 4 occurred in 59% of patients leading to discontinuation of therapy in 24.5% of patients and one death. Therapy with checkpoint inhibitors could be safer and more effective in combination with hyperthermia and fever inducing therapies. We conducted a retrospective analysis to test the safety and efficacy of a new combination immune therapy in 131 unselected stage IV solid cancer patients with 23 different histological types of cancer who exhausted all conventional treatments. Treatment consisted of locoregional- and whole-body hyperthermia, individually dose adapted interleukin 2 (IL-2) combined with low-dose ipilimumab (0.3 mg/kg) plus nivolumab (0.5 mg/kg). The objective response rate (ORR) was 31.3%, progression-free survival (PFS) was 10 months, survival probabilities at 6 months was 86.7% (95% CI, 81.0-92.8%), at 9 months was 73.5% (95% CI, 66.2-81.7%), at 12 months was 66.5% (95% CI, 58.6-75.4%), while at 24 months survival was 36.6% (95% CI:28.2%; 47.3%). irAEs of World Health Organization (WHO) Toxicity Scale grade 1, 2, 3, and 4 were observed in 23.66%, 16.03%, 6.11%, and 2.29% of patients, respectively. Our results suggest that the irAEs profile of the combined treatment is safer than that of the established protocols without compromising efficacy.

Thymosin beta 15A (TMSB15A) is a predictor of chemotherapy response in triple-negative breast cancer.

BACKGROUND: Biomarkers predictive of pathological complete response (pCR) to neoadjuvant chemotherapy (NACT) of breast cancer are urgently needed. METHODS: Using a training/validation approach for detection of predictive biomarkers in HER2-negative breast cancer, pre-therapeutic core biopsies from four independent cohorts were investigated: Gene array data were analysed in fresh frozen samples of two cohorts (n=86 and n=55). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in formalin-fixed, paraffin-embedded (FFPE) samples from two neoadjuvant phase III trials (GeparTrio, n=212, and GeparQuattro, n=383). RESULTS: A strong predictive capacity of thymosin beta 15 (TMSB15A) gene expression was evident in both fresh frozen cohorts (P<0.0001; P<0.0042). In the GeparTrio FFPE training cohort, a significant linear correlation between TMSB15A expression and pCR was apparent in triple-negative breast cancer (TNBC) (n=61, P=0.040). A cutoff point was then defined that divided TNBC into a low and a high expression group (pCR rate 16.0% vs 47.2%). Both linear correlation of TMSB15A mRNA levels (P=0.017) and the pre-defined cutoff point were validated in 134 TNBC from GeparQuattro (pCR rate 36.8% vs 17.0%, P=0.020). No significant predictive capacity was observed in luminal carcinomas from GeparTrio and GeparQuattro. CONCLUSION: In TNBC, TMSB15A gene expression analysis might help to select patients with a high chance for pCR after NACT.

Long-term outcome after neoadjuvant radiochemotherapy in locally advanced noninflammatory breast cancer and predictive factors for a pathologic complete remission : results of a multivariate analysis.

BACKGROUND: An earlier published series of neoadjuvant radiochemotherapy (NRT-CHX) in locally advanced noninflammatory breast cancer (LABC) has now been updated with a follow-up of more than 15 years. Long-term outcome data and predictive factors for pathologic complete response (pCR) were analyzed. PATIENTS AND METHODS: During 1991-1998, 315 LABC patients (cT1-cT4/cN0-N1) were treated with NRT-CHX. Preoperative radiotherapy (RT) consisted of external beam radiation therapy (EBRT) of 50 Gy (5 × 2 Gy/week) to the breast and the supra-/infraclavicular lymph nodes combined with an electron boost in 214 cases afterwards or-in case of breast conservation-a 10-Gy interstitial boost with (192)Ir afterloading before EBRT. Chemotherapy was administered prior to RT in 192 patients, and concomitantly in 113; 10 patients received no chemotherapy. The update of all follow-up ended in November 2011. Age, tumor grade, nodal status, hormone receptor status, simultaneous vs. sequential CHX, and the time interval between end of RT and surgery were examined in multivariate terms with pCR and overall survival as end point. RESULTS: The total pCR rate after neoadjuvant RT-CHX reached 29.2%, with LABC breast conservation becoming possible in 50.8% of cases. In initially node-positive cases (cN+), a complete nodal response (pN0) after NRT-CHX was observed in 56% (89/159). The multivariate analysis revealed that a longer time interval to surgery increased the probability for a pCR (HR 1.17 [95% CI 1.05-1.31], p < 0.01). However, in large tumors (T3-T4) a significantly reduced pCR rate (HR 0.89 [95% CI 0.80-0.99], p = 0.03) was obtained. Importantly, pCR was the strongest prognostic factor for long-term survival (HR 0.28 [95% CI 0.19-0.56], p < 0.001). CONCLUSION: pCR identifies patients with a significantly better prognosis for long-term survival. However, a long time interval to surgery (> 2 months) increases the probability of pCR after NRT-CHX.

Do insulin-like growth factor associated proteins qualify as a tumor marker? Results of a prospective study in 163 cancer patients.

OBJECTIVE: Insulin-like growth factor (IGF)-1, -2 and Insulin like growth factor binding proteins (IGFBP) are involved in the proliferation and differentiation of cells. It has never been evaluated, if the IGF-system can serve as a tumor marker in neoplasms. METHODS: In our prospective study 163 patients with colorectal cancer (22), prostate cancer (21), head and neck tumors (17), lymphomas (20), lung cancer (34) and other entities (49) were analysed for their IGF and IGFBP serum levels at the beginning and the end of radiotherapy and compared to 13 healthy people. Subgroups of patients with local tumor disease versus metastatic disease, primary and recurrent therapy and curative versus palliative therapy were compared. RESULTS: The serum levels of IGF-2 were significantly elevated in patients with prostate and colorectal cancer. However, sensitivity and specificity were only 70%. IGFBP-2 serum levels were elevated in patients with head and neck tumors. Again sensitivity and specificity were only 73%. A difference between local disease and metastatic disease could not be found. A difference between IGF serum levels before and after radiotherapy could not be detected. CONCLUSION: The IGF-system cannot serve as a new tumor marker. The detected differences are very small, sensitivity and specificity are too low. IGF measurement is not useful for the evaluation of the success of radiotherapy in malignancies.

Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease.

BACKGROUND: Tumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis. MATERIALS AND METHODS: We prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF. RESULTS: A hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients was detected. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001). CONCLUSION: The detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.

Gene expression of circulating tumour cells and its correlation with tumour stage in breast cancer patients.

BACKGROUND: Breast cancer (BC) represents one of the leading causes of cancer related deaths worldwide. New tools for diagnostic staging and therapeutic monitoring are needed to improve individualized therapies and improve clinical outcome. The analyses of circulating tumour cells may provide important prognostic information in the clinical setting. MATERIALS AND METHODS: Circulating tumour cells (CTC) of 63 BC patients were isolated from peripheral blood (PB) through immunomagnetic separation. Subsequently, RT-PCR or mPCR for the genes ga733.2, muc-1, c-erbB2, mgb-1, spdef and c-erbB2 were performed. Subsequently, expression data were correlated with the tumour stages. Fourteen healthy individuals served as controls. RESULTS: Significant correlations with tumour stages were found in single gene analyses of ga733.2, muc-1 and in multi-gene analyses of ga733.2/muc-1/mgb1/ spdef. Furthermore, a significant correlation of Ca 15-3 and all studied genes was also observed. CONCLUSION: Herein, we demonstrated a positive correlation of a gene signature consisting of ga733.2, muc-1, mgb1 and spdef and advanced stages of BC. Moreover, all studied genes and gene patterns revealed a significant correlation with Ca 15-3 positive cases.

Gene expression profiles in the acutely dissected human aorta.

OBJECTIVES: heritable connective tissue abnormalities and arterial hypertension may predispose to aortic dissection. This study evaluates gene expression profiles in the acutely dissected human aorta. DESIGN, MATERIALS AND METHODS: Atlas Human Broad Arrays I, II, and III (Clontech) were used to compare gene expression in acutely dissected (6 patients) and normal ascending aortas (6 multiorgan donors). The tissues were also compared macroscopically. RESULTS: of 3537 genes analysed, 1250 (35%) were expressed in aortic tissue. For statistical analysis we focused on 627 genes, which had an intensity>0.95 of the mean patients or controls. Dissected and adjacent macroscopically intact aorta displayed similar gene expression patterns. On the contrary, 66 genes were expressed significantly different in dissected aorta, compared with undiseased control aorta of multiorgan donors. Genes, predominantly upregulated in dissection, are involved in inflammation, in extracellular matrix proteolysis, in proliferation, translation and transcription. Predominantly downregulated genes code for extracellular matrix proteins, adhesion proteins and cytoskeleton proteins. CONCLUSION: our results demonstrate for the first time the complexity of the dissecting process on a molecular level. The ultimate dissection seems to be the dramatic endpoint of a long-lasting process of degradation and insufficient remodelling of the aortic wall. Altered patterns of gene expression suggest a pre-existing structural failure of the aortic wall, resulting in dissection.

Novel lipophilic chloroquine analogues for a highly efficient gene transfer into gynecological tumors.

Liposomal vectors based on cationic lipids have been proven to be an attractive alternative to viral vectors in gene therapy protocols with regard to safety and manufacturing concerns. In order to improve the transfection efficiency we have synthesized two novel carboxycholesteryl-modified chloroquine analogues. Due to their potential endosomal buffering capacity these compounds enable the efficient transfection of various gynecological tumors and therefore are promising reagents in gene therapy applications.

Long-term prognosis of breast cancer patients with 10 or more positive lymph nodes treated with CMF.

The purpose of this investigation was to study the long-term prognosis of breast cancer patients with 10 or more positive lymph nodes after conventional chemotherapy treatment with cyclophosphamide, methotrexate and 5-fluorouracil (CMF). Between 1984 and 1989, 1048 node-positive patients were treated with CMF in two separate trials conducted by the German Breast Cancer Study Group (GBSG). Subgroups either received radiotherapy or tamoxifen in addition. In this study, long-term prognosis in the subgroup of 141 patients with 10 or more positive lymph nodes was investigated. Univariate and multivariate Cox models were used to evaluate the effect of prognostic factors on event-free survival (EFS) and overall survival (OS). Both univariate and multivariate analyses revealed the progesterone receptor (PR) status as the dominating prognostic factor for both EFS and OS, resulting in a strongly increased risk of more than 2-fold for receptor-negative patients. A large number of positive lymph nodes also affected the prognosis for EFS. In univariate analysis, the degree of lymph node involvement (i.e. percentage of positive nodes out of all examined nodes), oestrogen status (ER) status, and tumour grade also showed significant effects. To conclude, the prognosis in the subgroup of patients with 10 or more positive lymph nodes is heterogeneous. Some surprisingly high survival rates have been observed in case series of breast cancer patients treated with high-dose chemotherapy which may be explained by patient selection. From the usual factors investigated in this study, the PR status showed the strongest effect, and, at least this factor should be taken into account in the design and analysis of trials for breast cancer patients with a poor prognosis.

Local inflammation and devascularization--in vivo mechanisms of the "bystander effect" in VPC-mediated HSV-Tk/GCV gene therapy for human malignant glioma.

Somatic gene therapy with the herpes simplex virus type I thymidine kinase gene/ganciclovir (HSV-Tk/GCV) system and murine retroviral vector producer cells (VPCs) was introduced as a new adjuvant treatment modality to treat tumor bulk and to prevent tumor recurrence in patients harboring malignant glioma. The single-center experience after treatment of 27 patients undergoing tumor resection followed by intracerebral VPC injection for HSV-Tk suicide gene therapy will be presented focused on findings of systematic and close MRI follow-up and a few histological specimens. The data indicate that hemorrhagic necrosis due to endothelial cell transfection mediated vessel necrosis and that local inflammatory immune response occurs frequently after gene therapy. These phenomena seem to be specific because none of the patients of a control group showed any similar features. The prognosis (time to progression, survival) of the patients with "bystander effects" after gene therapy was better, but compared to those patients without bystander effects, they were also privileged by a favorable constellation of prognostic factors. Therefore, the appearance of these neuroradiologic features cannot serve as an indicator for treatment effectiveness and outcome.

Randomized trial on the effect of radiotherapy in addition to 6 cycles CMF in node-positive breast-cancer patients. The German Breast-Cancer Study Group.

In 1984 the GBSG started a multicenter randomized trial to compare the effectiveness of 6 cycles of cyclophosphamide, methotrexate and fluorouracil (CMF) with or without radiotherapy (RT) as adjuvant treatment in node-positive breast-cancer patients treated by mastectomy. During 5 years, 199 patients were randomized. After a median follow-up of about 9 years, the treatment groups 6 x CMF and 6 x CMF + RT were compared regarding time to recurrence and death. As the first event of failure, we observed locoregional recurrence in 22 patients, distant metastases in 66 patients, a secondary malignancy in 9 patients and death without previous event in 5 patients. For event-free survival (EFS), no significant difference was observed [relative risk (RR) 6 x CMF + RT vs. 6 x CMF 0.82, 95% confidence interval (CI) 0.55-1.21]. Event-specific analysis showed a significant decreased risk after radiotherapy for locoregional recurrence. The risk for distant metastases was estimated as slightly decreased and the risk for secondary malignancy and for death without previous event was estimated as increased in treatment group 6 x CMF + RT in comparison with treatment group 6 x CMF, but these effects were not significant. For overall survival (OS) and breast-cancer-specific survival (BCS), no significant treatment effect could be demonstrated. There is a beneficial effect of radiotherapy on locoregional recurrence. For EFS and BCS, a tendency in favour of radiotherapy is observed, but this is not significant; for OS, no difference can be demonstrated, but the power of the study is too low to detect small treatment effects.

Randomized 2 x 2 trial evaluating hormonal treatment and the duration of chemotherapy in node-positive breast cancer patients: an update based on 10 years' follow-up. German Breast Cancer Study Group.

PURPOSE: In 1984, the German Breast Cancer Study Group started a multicenter randomized trial to compare six versus three cycles of cyclophosphamide, methotrexate, and fluorouracil (CMF) starting perioperatively and to investigate the additional effect of tamoxifen as adjuvant treatment in node-positive breast cancer patients treated with mastectomy. PATIENTS AND METHODS: From 1984 to 1989, 473 patients were randomized from 41 institutions. After a median follow-up of approximately 10 years for overall survival (OS) and 9 years for event-free survival (EFS), the treatment groups were compared with respect to OS and EFS. Results based on a median follow-up of 56 months have been published earlier. RESULTS: Estimated cumulative locoregional incidence rate after 10 years was 19.9%; the corresponding rate of distant recurrences was 41.3%. Concerning duration of chemotherapy, we did not find any difference between six and three cycles of CMF (EFS: relative risk [RR] in multivariate analysis = 0.95; 95% confidence interval [CI], 0.74 to 1.21 OS: RR = 0.90; 95% CI, = 0.69 to 1.18). Treatment with tamoxifen resulted in an improvement in outcome (EFS: RR = 0.81; 95% CI, 0.61 to 1.07, OS: RR = 0.74; 95% CI, 0.55 to 1.0) although it proved not significant. Number of positive lymph nodes and progesterone receptor were the dominant prognostic factors. CONCLUSION: In this study, we observed some tendency in favor of hormonal treatment, which is in agreement with the literature. Concerning duration of chemotherapy, the results of this study provide further evidence that a reduction to three cycles of CMF is possible without increasing the risk of recurrence or death. For a definitive conclusion, however, further investigations are required.

Gene Therapy for Treatment of Brain Tumors (HSV-tKIn Vivo Gene Transfer) : A Case Study.

Despite a high effort in the research of malignant brain tumors, the clinical results in treatment of malignant brain tumors are still very poor. Brain tumors are a major cause of morbidity and mortality in the population. New primary brain tumors develop in 2-4 of 100,000 adults each year (1). Recent evidence indicates that the prevalence of primary brain tumors is increasing, especially in the elderly (2). The astroglial brain tumors, including the highly malignant glioblastoma multiforme (GBM), are the most common primary brain tumors. For these tumors, the first line of treatment is surgery and almost always radiotherapy as an adjuvant. A variety of patient-management strategies are currently used for GBM, from supportive care to aggressive multimodality approaches. The principal reason for this wide spectrum of approaches is that, despite aggressive therapy, which includes surgical removal of the tumor, postoperative high-dose radiation (60 gy), chemotherapy, and other adjuvant treatments, the prognosis of patients with GBM is very poor (3 -6). In a series of NCOG protocols on glioblastoma multiforme patients with Karnofsky performance scores of 60 or higher, who were treated with postsurgical radiation therapy and adjuvant chemotherapy with nitrosourea-based drug combinations, the median survival and time of tumor progression were consistently above 50 and 34 wk, respectively (7 -9). The nitrosoureas (BCNU and CCNU), alone and in combination, are the most active cytotoxic drugs for recurrent and progressive tumors, although most of these responses are transient and in patients with well-differentiated gliomas. When glioblastoma multiforme recurs, which happens in nearly 100% of all cases, however, the median survival from the start of treatment is about 6 mo, with only 22% of patients surviving longer than 1 yr (11). Therefore, there is a great interest in local treatment modalities. A wafer impregnated with carmustine, for use as an implant after surgical removal of recurrent GBM showed a prolongation in the median survival time of only 2 mo, from 20 to 28 wk in a study with a total of 222 patients. In another study, a median survival of 9 mo was found in a selected group of patients with recurrent GBM who underwent a second operation, but a reasonable quality of life in those patients was limited to 10 wk (12).

Modelling the effects of standard prognostic factors in node-positive breast cancer. German Breast Cancer Study Group (GBSG).

Prognostic models that predict the clinical course of a breast cancer patient are important in oncology. We propose an approach to constructing such models based on fractional polynomials in which useful transformations of the continuous factors are determined. The idea may be applied with all types of regression model, including Cox regression, the method of choice for survival-time data. We analyse a prospective study of node-positive breast cancer. Seven standard prognostic factors--age, menopausal status, tumour size, tumour grade, number of positive lymph nodes, progesterone and oestrogen receptor concentrations--were investigated in 686 patients, of whom 299 had an event for recurrence-free survival and 171 died. We determine a final model with transformations of prognostic factors and compare it with the more traditional approaches using categorized variables or assuming a straight line relationship. We conclude that analysis using fractional polynomials can extract important prognostic information which the traditional approaches may miss.

Dual-parameter immunoflow cytometry in diagnosis and follow-up of patients with bladder cancer.

OBJECTIVES: The aim of this study was to evaluate the clinical significance of a dual-parameter immunoflow cytometry (DPI-FCM) assay in the detection of tumor cells in barbotage specimens of bladder cancer patients. METHODS: DPI-FCM is an automated method, based on the utilization of two monoclonal antibodies (mAbs) either used for a preselection of urothelial cells (mAb Due AUT 2) or the analysis of the expression of a differentiation antigen within the preselected urothelial cells (mAb Due ABC 3). The ratio of ABC 3-positive urothelial cells was used to discriminate between the normal and malignant state of the urothelium. At the time of examination 40 patients had endoscopically overt bladder tumors. Another 30 patients without endoscopically visible tumors were examined before routine rebiopsy. Thirty barbotage specimens from patients with diseases not related to bladder cancer were examined as controls. RESULTS: Overall, the sensitivity of DPI-FCM in patients with endoscopically overt and invisible residual tumors was 95 and 83%, respectively, regardless of concomitant urinary tract infection. The sensitivity of DPI-FCM for both patient groups was 86, 95 and 94% for tumor grades 1, 2 and 3, respectively. The specificity of the method in 30 patients with no history of bladder cancer was 93%. CONCLUSIONS: DPI-FCM appears to be a highly reliable method of recognizing tumor cells in bladder barbotage specimens even in patients with concomitant urinary tract infection. The procedure may be of value in monitoring bladder cancer patients for tumor recurrence.

The effect of oral contraceptive use on the prognosis of node positive breast cancer patients. German Breast Cancer Study Group.

The effect of oral contraceptive (OC) use as a risk factor for breast cancer was recently assessed in a large meta-analysis, but currently available data on the prognostic effect are still insufficient. We investigated the relationship between OC use and standard prognostic factors and the effect of OC use on recurrence-free survival (RFS) and overall survival (OS) in 422 premenopausal pT1a-3aN + M0 patients from two trials of the German Breast Cancer Study Group (GBSG). 137 patients (32.5%) were OC users. They were younger on average (mean age 41.5 years versus 45 years for non-OC users) and the percentage of patients with smaller tumours was higher in the group of OC users. Based on 163 events for RFS and 103 events for OS, no significant effect of OC use on RFS and OS could be demonstrated in univariate and multivariate analyses. In our study of node positive breast cancer cases, OC users were younger and had smaller tumours. This may be an effect of earlier detection of breast cancer, but OC users did not have a better prognosis, both before and after adjustment for tumour size and other prognostic factors.

Grading system for breast cancer.

As recurrence rates in breast cancer depend significantly on degree of malignancy (6-9) and mitotic rates as part of the grading system are difficult to assess in exact manner, we widened the grading system by evaluation of the proliferative compartment to get more information about proliferation activity as an important factor for tumor-progression (15, 16). This additional analysis can be used as a control factor for the correctness of the evaluation of mitotic activity. We practice this procedure at present as a "working formulation". New antibodies are in preparation which allow to exclude the highly variable G1-Phase. Further precision and control of degree of malignancy can be achieved by photometric analysis of S-Phase, 5c exceeding rate and state of ploidy according to Auer scheme.

WAF1/p21 regulates proliferation, but does not mediate p53-dependent apoptosis in urothelial carcinoma cell lines.

The WAF1/p21 gene product is an inhibitor of cyclin-dependent kinases which can be induced by the tumor suppressor p53 and mediate some of its effects, or function in p53-independent pathways of cell cycle regulation. Although a potential tumor suppressor gene, WAF1/p21 is expressed in bladder cancer. To elucidate the function of p21 in tumor cells we have investigated in urothelial carcinoma cell lines: i) WAF1/p21 mRNA and protein expression, ii) the biological effects of p21 overexpression or down-regulation and (iii) whether p21 can be induced by p53. WAF1/p21 mRNA levels examined in four cell lines were comparable to bladder mucosa. One cell line, HT1376, failed to express p21 protein due to a frame shift mutation. Overexpression of WAF1/p21 cDNA inhibited clone formation in three cell lines, whereas transfection with antisense WAF1 increased clone sizes and numbers. WAF1 sense clones showed diminished cell proliferation compared to the parental cell line. Apoptosis- induced wild-type p53 was not inhibited by overexpression of antisense WAF1/p21. In a cell clone derived from line VMCub1 by stable transfection with wild-type p53 under the control of a metallothionein promotor, p21 was induced along with p53 upon activation of the promoter with zinc chloride. This induction was accompanied by a decrease in cell proliferation but by little apoptosis. These data suggest that p21 inhibits proliferation in a p53-dependent or independent manner but does not mediate p53-induced apoptosis in urothelial carcinoma cells.

Effect of recombinant human granulocyte colony stimulating factor (R-metHuG-CSF) as an adjunct to large-field radiotherapy: a phase I study.

PURPOSE: To test the feasibility of recombinant human granulocyte colony stimulating factor application during large-field radiotherapy. METHODS AND MATERIALS: Fifteen patients with clinically and histologically proven malignancy who received large-field radiotherapy entered this study. Administration of recombinant granulocyte colony stimulating factor (G-CSF) at a dose of 300 microgram subcutaneously was started on Friday and was continued on Saturday and Sunday after the first radiotherapy treatment, which began on the Monday before. In this way four courses of G-CSF were applied every Friday, Saturday, and Sunday during the radiotherapy period. Absolute neutrophil cell (ANC) and blood counts were monitored twice a week and compared to a second group of 15 patients who received large-field radiotherapy without G-CSF. Before and at the end of every cycle of G-CSF, ANC, blood counts, and biochemistry were measured. We compared the myelotoxicity of the patients treated with G-CSF with 15 patients without G-CSF treated at the same period with large-field radiotherapy, in match pair technique. RESULTS: G-CSF increased the ANC throughout the period of irradiation, and the treatment time needed for competing radiotherapy was shorter in the group who received G-CSF. Fourteen of 15 patients who received G-CSF treatment completed large-field radiotherapy without pause. Only 1 of 15 patients not receiving G-CSF was able to receive radiation treatment on schedule. Patients receiving G-CSF completed treatment with the mantle-field technique in 24 days and those with the abdominal bath technique in 26.5 days. Conversely, patients treated without G-CSF completed treatment with the mantle-field technique in 30.5 days and those with the abdominal bath technique in 36 days. The most frequent side effect was musculoskeletal pain. CONCLUSION: The prophylactic application of G-CSF during large-field radiotherapy before the onset of neutropenia was feasible in this schedule. Whether or not this shortening of treatment duration will translate into an improvement in efficacy is not clear.