Nicholas H. A. Terry

Can we detect or predict the presence of occult nodal metastases in patients with squamous carcinoma of the oral tongue

When to do a neck dissection as part of the surgical treatment for a patient with squamous carcinoma of the oral tongue is controversial, particularly when the primary can be resected without entering the neck. If the patient who is at high risk for having occult nodal disease in the neck can be identified, node dissection with the glossectomy could be justified. To better identify patients for this procedure, we correlated various tumor and patient factors along with preoperative diagnostic studies with the presence or absence of pathologically positive nodes in a group of patients who underwent node dissection.

Supraadditive apoptotic response of R3327-G rat prostate tumors to androgen ablation and radiation

PURPOSE Androgen ablation is often combined with radiation in the treatment of patients with prostate cancer, yet, the optimal sequencing and the mechanisms governing the interaction are not understood. The objectives were to determine if cell killing via apoptosis is enhanced when the combined treatment is administered and to define the relationship of changes in this form of cell killing to tumor volume growth delay. MATERIALS AND METHODS Dunning R3327-G rat prostate tumors, grown in the flanks of Copenhagen rats, were used at a volume of approximately 1 cc. Androgen ablation was initiated by castration, and androgen restoration was achieved with 0.5 cm silastic tube implants containing testosterone. 60Co was used for irradiation. The terminal deoxynucleotidyl transferase (TUNEL) histochemical assay was used to quantify apoptosis. RESULTS Tumors from intact and castrate unirradiated control rats had average apoptotic indices (percent of apoptotic cells) of 0.4 and 1.0%, respectively. The apoptotic index varied only slightly over time (3 h to 28 days) after castration (range 0.75-1.43%). Irradiation of intact rats to 7 Gy resulted in a peak apoptotic response at 6 h of 2.3%. A supraadditive apoptotic response was seen when castration was initiated 3 days prior to 7 Gy radiation, with peak levels of about 10.1%. When the radiation was administered at increasing times beyond 3 days after castration, the apoptotic response gradually diminished and was back to levels seen in intact rats by 28 days after castration. Tumor volume growth delay studies were consistent with, but not conclusive proof of, a supraadditive effect when the combination was used. DISCUSSION A supraadditive apoptotic response was seen when androgen ablation and radiation were used to treat androgen sensitive R3327-G rat prostate tumors. This supraadditive effect was dependent on the timing of the two treatments. Further studies are required to more fully define the optimal timing and administration of androgen ablation and radiation.

Resistance of Differentiating Spermatogonia to Radiation-Induced Apoptosis and Loss in p53-Deficient Mice

The effect of the p53 gene on the survival of mouse testicular cells was evaluated by analysis of degenerating and terminal transferase-mediated end labeling (TUNEL)-positive cells and the subsequent production of further differentiated progeny. In p53 null mice, in contrast to wild-type mice, radiation induced negligible levels of degenerating or TUNEL-positive differentiating spermatogonia within 24 h. This was correlated with higher production of differentiated progeny of the differentiating spermatogonia in p53 null mice. Contrary to the differentiating spermatogonia, the stem spermatogonia of p53 null mice produced fewer differentiated progeny after irradiation than did the stem cells of wild-type mice. We conclude that, because the degeneration and TUNEL positivity of the differentiating spermatogonia in mice of different genotypes were correlated with each other and were dependent on p53, this process is indeed apoptosis. In the differentiating spermatogonia, p53-dependent apoptosis accounted for the bulk of the loss of their progeny after irradiation. Furthermore, whereas the differentiating spermatogonia died by apoptosis that was dependent on p53, the stem spermatogonia, which are more radioresistant, did not.

A CD40 Signalosome anchored in lipid rafts leads to constitutive activation of NF-κB and autonomous cell growth in B cell lymphomas

B cell lineage non-Hodgkins lymphomas (NHL-B) are neoplastic B cells that show dysregulated B lymphocyte growth characteristics. Unlike normal B cells, aggressive NHL-B cells show constitutive expression of nuclear NF-kappaB by maintaining an assembled, scaffold-like signaling platform, called a Signalosome within the lipid raft microdomain, extending from the cell membrane. The CD40 Signalosome appears to be initiated through autochthonous production and cognate binding of CD154 (CD40L, gp39) to CD40 by the lymphoma cell. Constitutive expression of NF-kappaB in NHL-B can be downregulated by treatment with antibodies to CD40 or CD154 that disrupt Signalosomes, inhibit lymphoma cell growth, and induce cell death. CD40 Signalosomes may provide a potentially vulnerable target for therapeutic intervention in NHL-B cells.

APOPTOSIS AND DOWNSTAGING AFTER PREOPERATIVE RADIOTHERAPY FOR MUSCLE-INVASIVE BLADDER CANCER

PURPOSE To determine the relationship between pretreatment apoptosis levels and clinical-to-pathologic downstaging resulting from preoperative radiotherapy. METHODS AND MATERIALS Between 1960-1983, 338 patients were dispositioned to receive preoperative radiotherapy 4-6 weeks prior to radical cystectomy for muscle-invasive transitional cell carcinoma of the bladder. Of these, adequate hematoxylin and eosin stained tissue sections for morphologic analysis of apoptosis were available in 158 patients. These patients were treated to a median dose of 50 Gy at 2 Gy per fraction. Median follow-up was 90 months. The apoptotic index (AI) was calculated from the ratio of the number of apoptotic cells divided by the total counted and multiplied by 100. A minimum of 500 cells were counted from each patient. RESULTS The average AI for the whole group (n = 158) was 2.0 +/- 1.3 (+/- SD), with a median of 1.8. The association of AI to clinical stage was significant with AI averages of 1.8 for Stage T2 (n = 56), 1.9 for T3a (n = 51), and 2.4 for T3b (p = 0.038, Kendall Correlation). The relationship of AI to radiotherapy response also was significant with an average of 2.2 for those who were downstaged (n = 103), 1.9 for those in whom the stage remained unchanged (n = 20), and 1.7 for those who were upstaged (n = 35, p = 0.054, Kendall Correlation). The other significant correlations with AI were for the factors, grade, mitotic index, number of tumors, and gender. The AI was then categorized into three groups ( < or = 1, > 1, and < or = 3, and > 3) to examine the prognostic significance of this parameter. The distributions of patients by clinical stage, grade, mitotic index, number of tumors, radiotherapy response, and hemoglobin level were significantly associated with AI using this grouping. When the analysis of the distribution of patients by radiation response and AI was segregated by stage, a significant correlation was observed only for those with Stage T3b disease (p = 0.006); 93% of T3b patients with an AI > 3 were downstaged, while in 7% the stage remained unchanged and none were upstaged. The relationship of AI to 5-year actuarial patient outcome was investigated using several end points and although no significant correlations were observed, a trend was seen for improved survival when AI was > 3 (71% vs. 41%, p = 0.09) for Stage T3b patients. CONCLUSION The AI correlated most strongly with radiotherapy response for patients with clinical stage T3b disease, the one subgroup of patients wherein preoperative radiotherapy is likely to be of the most benefit. Further investigation of pretreatment apoptosis levels as a marker of anticancer response is needed, especially for patients treated with chemotherapy and radiotherapy with the goal of bladder preservation.

Enhanced radioresponse of paclitaxel-sensitive and -resistant tumours in vivo

Paclitaxel is a potent chemotherapeutic drug and also has the potential to act as a radioenhancing agent. The latter is based on its ability to arrest cells in the radiosensitive G2M phases of the cell cycle; the weight of supporting evidence is derived mainly from in vitro studies. Our previous in vivo experiments identified enhanced tumour radioresponse predominantly attributable to tumour reoxygenation occurring as a result of paclitaxel-induced apoptosis. The current study investigated whether paclitaxel enhanced the radioresponse of tumours which are insensitive to apoptosis induction, but exhibited mitotic arrest, and compared the degree and kinetics of the response to that in tumours which develop apoptosis. The mouse mammary carcinoma MCa-29 (apoptosis sensitive) and the squamous cell carcinoma SCC-VII (apoptosis resistant) were used. In addition, the study investigated whether paclitaxel affected normal skin radioresponse to determine if a therapeutic gain could be achieved. Paclitaxel enhanced the radioresponse of both types of tumours. In the SCC-VII tumour, radiopotentiation occurred within 12 h of paclitaxel administration coincident with mitotic arrest, where enhancement factors (EFs) ranged from 1.15 to 1.37. In MCa-29 tumour, the effect was greater, EFs ranging from 1.59 to 1.91 and occurred between 24 and 72 h after paclitaxel when apoptosis was the predominant microscopic feature of treated tumours and when tumour oxygenation was found to be increased. The acute skin radioresponse and late leg contracture response were essentially unaffected by prior treatment with paclitaxel. Therefore, by two distinct mechanisms, paclitaxel was able to enhance the radioresponse of paclitaxel-sensitive and -resistant tumours, but not the normal tissue radioresponse, thus providing true therapeutic gain.

Relationship of Ki-67 labeling index to DNA-ploidy, S-phase fraction, and outcome in prostate cancer treated with radiotherapy

Our purpose was to evaluate the relationship of Ki‐67 labeling index (Ki67‐LI) to deoxyribonucleic acid (DNA) ploidy, S phase fraction (SPF), other clinical prognostic factors, and clinical outcome for patients with prostate cancer treated by external beam radiotherapy.

Flow cytometry after bromodeoxyuridine labeling to measure S and G2+M phase durations plus doubling times in vitro and in vivo

This protocol describes methods for calculating the proliferative parameters of cell populations. The basis of the technique is to label cells, either in vitro or in vivo, with halogenated thymidine analogs, such as bromodeoxyuridine (BrdU). Bivariate DNA-BrdU flow cytometry is used to analyze the BrdU-labeled and unlabeled cells. The enumeration of specific cohorts of cells that either have or have not divided in the interval between labeling and cell/tissue sampling permits the calculation of the potential doubling time (Tpot) of the population, plus the durations of DNA synthesis (TS) and the G2+M phase (TG2+M) of the cell cycle. The method provides information that is not otherwise available, namely inhibition of DNA synthesis and the separate evaluation of cell-cycle effects in BrdU-labeled and unlabeled subpopulations. Ethanol-fixed samples take 1 d to prepare and stain, and reliable parameter estimates might be obtained from measurements made at a single time point after labeling.

Comparative Effect of the Thiols Dithiothreitol, Cysteamine and WR-151326 on Survival and on the Induction of DNA Damage in Cultured Chinese Hamster Ovary Cells Exposed to γ-radiation

We compared the ability of three thiols--dithiothreitol (DTT), cysteamine and WR-151326--to protect aerated Chinese hamster ovary cells from the lethal and DNA-damaging effects of gamma-radiation. These results were compared with earlier measurements for WR-1065 and WR-255591. The time-course and the concentration dependence of protection against cell killing was determined after 10 Gy of gamma-rays. The aminothiols cysteamine and WR-151326 protected at much lower extracellular concentrations than the simple thiol DTT; however, there was no clear difference between the behaviour of cysteamine, WR-151326, WR-1065 and WR-255591 in this respect. Protection by DTT and cysteamine was complete within 1 min, whereas for WR-151326, WR-1065 and WR-255591 about 30 min was required before protection began to reach a plateau. Based on these data, complete radiation survival curves were generated for each thiol and protection factors calculated. Effects on the induction of DNA single-strand breaks (ssb) and double-strand breaks (dsb) by gamma-rays were measured using alkaline (pH 12.1) and neutral (pH 7.0 and 9.6) elution, respectively. All three thiols protected against ssb induction, although to a significantly lower extent than against cell killing measured under identical conditions. Each thiol also protected against dsb induction. After high radiation doses the protection factors for dsb induction were also less than the protection factors for cell survival; however, when dsb were assayed using the low-dose replicate plating neutral elution method, the relative effect of each thiol on cell survival and on dsb induction appeared to be equivalent. The hierarchy of protection against both ssb and dsb induction (based on the extracellular thiol concentration required to produce a given degree of protection) was similar to that for cell survival, i.e., WR-151326 congruent to cysteamine less than DTT.

Relationship of tumor DNA-ploidy toserum prostate-specific antigen doubling time after radiotherapy for prostate cancer

OBJECTIVES DNA-ploidy is a strong prognostic factor for prostate cancer patients treated with definitive external beam radiotherapy. Using DNA/nuclear protein flow cytometry, three prognostic groups based on DNA-ploidy were identified: from good to poor, these are diploid, near-diploid, and nondiploid tumors. Since recent evidence indicates that the rate at which prostate-specific antigen (PSA) increases in the presence of biochemical failure is predictive of the time to clinical relapse, we examined the relationship between DNA-ploidy and PSA doubling time (PSA-DT). METHODS Formalin-fixed paraffin-embedded tissues from 76 patients treated at M.D. Anderson Cancer Center with definitive radiotherapy alone were analyzed for ploidy using DNA/nuclear protein flow cytometry. Of these, 24 of the 27 patients with a rising PSA profile had three or more post-treatment PSA values from which the PSA-DTs were calculated. PSA-DTs were estimated using nonlinear regression techniques. RESULTS The average PSA-DT for the 24 patients in this cohort was 11.3 +/- 10.5 months (+/- SD) with a median of 8.4 months. Diploidy (n = 3) was associated with a PSA-DT of 27.0 +/- 22.8 months, near-diploidy (n = 7) with a PSA-DT of 12.2 +/- 5.7 months, and non-diploidy (n = 14) with a PSA-DT of 7.5 +/- 5.7 months (p = 0.004, Spearman rank test). Stage, grade, and pretreatment PSA, as well as the endpoints of local control, freedom from metastases, and freedom from any relapse, did not correlate significantly with PSA-DT values. However, when patients were subdivided by PSA-DT into those with values 10 months or less (n = 14) and those more than 10 months (n = 10), there was a correlation with 3-year actuarial freedom from relapse: 28% and 74%, respectively (p < 0.01, log-rank). This subdivision of PSA-DT also correlated with DNA-ploidy (p = 0.03, chi-square) and stage (p = 0.04). CONCLUSIONS The results show that there is a significant correlation of DNA-ploidy with PSA-DT. Diploidy was associated with the longest PSA-DTs, near-diploidy with intermediate PSA-DTs, and nondiploidy with short PSA-DTs. Patients with short PSA-DTs also had significantly higher actuarial rates of disease relapse at 3 years. These data confirm that PSA-DT is a strong predictor of tumor behavior and that patients who have nondiploid tumors probably require more aggressive, combined modality, treatment.