Screening of five specific cell cycle inhibitors using a T cell lymphoma cell line synchrony/release assay.

To obtain different cell populations at specific cell cycle stages, we used a cell culture synchronization protocol. Effects of five different cell cycle inhibitors acting throughout the cell cycle were examined by DNA flow cytometric analysis of a synchrony/release lymphoma cell line (CEM). The screening synchronized protocol showed that staurosporine, mimosine and aphidicolin are reversible G1 phase inhibitors that act at different times. Staurosporine acted in early G1, exhibited the strongest cytotoxic effect, and induced apoptosis. Mimosine and aphidicolin acted in late G1 and at the G1/S boundary, respectively. Hydroxyurea arrested CEM cells in early S phase, but later than the aphidicolin arrest point. Nocodazole synchronized CEM cells in M phase. All the inhibitors examined in this study can be used to synchronize cells at different phases of the cell cycle and were reversible with little toxicity except for staurosporine which is highly toxic. Because the regulatory mechanism of the cell cycle is disrupted by their effects on protein synthesis, however, these drugs must be used with caution.

Reproducibility of measurements of potential doubling time of tumour cells in the multicentre National Cancer Institute protocol T92-0045.

We compared the flow cytometric measurement and analysis of the potential doubling time (Tpot) between three centres involved in the National Cancer Institute (NCI) protocol T92-0045. The primary purpose was to understand and minimize the variation within the measurement. A total of 102 specimens were selected at random from patients entered into the trial. Samples were prepared, stained, run and analysed in each centre and a single set of data analysed by all three centres. Analysis of the disc data set revealed that the measurement of labelling index (LI) was robust and reproducible. The estimation of duration of S-phase (T(S)) was subject to errors of profile interpretation, particularly DNA ploidy status, and analysis. The LI dominated the variation in Tpot such that the level of final agreement, after removal of outliers and ploidy agreement, reached correlation coefficients of 0.9. The sample data showed poor agreement within each of the components of the measurement. There was some improvement when ploidy was in agreement, but correlation coefficients failed to exceed values of 0.5 for Tpot. The data suggest that observer-associated analysis of T(S) and tissue processing and tumour heterogeneity were the major causes of variability in the Tpot measurement. The first two aspects can be standardized and minimized, but heterogeneity will remain a problem with biopsy techniques.

Expression of cyclins and cdks throughout murine carcinogenesis.

The overexpression and/or amplification of cell cycle regulating genes is an important factor in the progression of cancer. Recent attention has been focused on several cyclin and cdks genes whose expression were increased in many types of tumor. In this study, we investigated the expression kinetics of cyclins A, B, D1, E and cdks 1, 2, 4, 6 by RT-PCR coupled with densitometry and correlated to the growth fraction (percentage of S cells). This analysis was performed using an experimental murine leukemic model, generated by in vivo administration of murine clonogenic cells Wehi-3b injected into balb-c mice. Differential expression of cyclins and cdks was observed between normal and tumoral cells with different patterns of expression between G1 and G2M cyclins-cdks. G1 cyclins cdks expression was significantly increased in tumor cells when compared to normal cells. In the same manner, G2M cyclins cdks expression was only observed in tumor cells at a lower level than for G1 cyclins cdks, but not detected in normal cells. These differences correlated with the growth fraction for both the G1 cyclins cdks (r = 0.91, 0.94, 0.85, 0.90 and 0.96 for cyclin D1, cyclin E, cdk2, cdk4 and cdk6, respectively) and the G2M cyclins cdks (r = 0.96, 0.97 and 0.93 for cyclins A, B and cdkl respectively). Analysis of cyclins cdks expression kinetics during tumoral progression shows that cyclins A, B and cdkl were expressed from the 12th day on of disease, increased until the death of the animals and correlated with the growth fraction (r = 0.94, 0.95 and 0.97 for cyclins A, B and cdk1 respectively) (n = 20). Overexpression of other cyclins cdks were observed, from the 6th day on for cyclin D1, the 12th day for cdk2 and cdk4, the 15th day for cdk6 and the 20th day for cyclin E. These increases persisted during tumoral progression and correlated with the growth fraction (r = 0.85, 0.94, 0.93, 0.96, and 0.98 for cyclin D1, cyclin E, cdk2, cdk4 and cdk6, respectively) (n = 20). Our results demonstrated that G1 and G2-M cyclins cdks mRNA levels were increased at approximately the same time of maximal tumor growth. Only cyclin D1 overexpression occured at the initiation of tumoral development, and could therefore be considered as an early marker of cell proliferation.

In vivo alloreactive potential of ex vivo-expanded primary T lymphocytes.

BACKGROUND: We are presently investigating the therapeutic potential of herpes simplex-thymidine kinase-expressing donor T cells in the setting of a T cell-depleted allogeneic bone marrow transplantation. The generation, expansion, and selection of the gene-modified T cells require a 12-day ex vivo culture period in high-dose interleukin (IL)-2 that could significantly alter their in vivo alloreactivity. METHODS: We evaluated the alloreactive potential of such cultured cells in a murine allogeneic bone marrow transplantation model. RESULTS: The present studies demonstrate that ex vivo-expanded cultured T cells are capable of strong alloreactivity as evidenced by the occurrence of lethal acute graft-versus-host disease (GVHD). However, GVHD mortality after administration of the cultured T cells occurred later than after the administration of a same number of fresh T cells. Similar kinetics of GVHD-induced mortality between cultured and fresh T cells required a 10-fold increase in the number of cultured T cells, indicating a reduced alloreactive potential of these cells. The addition of a 2-day "resting" period in low-dose IL-2 resulted in T cells with enhanced alloreactive potential identical to the alloreactivity observed with fresh T cells. CONCLUSION: Ex vivo IL-2-expanded T cells are capable of significant in vivo alloreactivity. However, an increase in the number of cultured T cells administered or the introduction of a short resting culture period prior to infusion is necessary in order to achieve in vivo alloreactivity identical to the alloreactivity observed with fresh T cells.

Differential effects of cyclosporin A on the alloreactivity of fresh and ex vivo-expanded T lymphocytes.

GVHD remains a major source of morbidity and mortality after non-T cell-depleted BMT. The use of donor T lymphocytes expressing a suicide gene could lead to specific immunomodulation after BMT. We are currently evaluating such an approach in a phase I clinical study. A 12-day ex vivo expansion is required to generate gene-modified donor T lymphocytes. CsA is commonly used for GVHD prophylaxis. We analyzed, in a murine GVHD model, the effects of CsA administration on the alloreactivity of fresh or ex vivo-expanded T cells. Variable amounts of fresh or ex vivo-expanded T cells were administered in conjunction with a marrow graft to lethally irradiated allogeneic mice. As expected, a protective effect of CsA with a delayed GVHD-related mortality (P < 0.01 vs saline treatment) was observed in mice receiving fresh splenocytes. However, CsA treatment had no effect (P = NS) in mice experiencing lethal GVHD induced by ex vivo-expanded T cells whether or not the T cells had been 'rested' in low-dose IL-2 prior to in vivo administration. In agreement with the in vivo findings, CsA also inhibited the in vitro proliferation of alloreactive fresh T cells while having no significant inhibitory effect on the alloreactivity of ex vivo-expanded T lymphocytes. Overall, we demonstrate that the alloreactivity of ex vivo-expanded T lymphocytes is not sensitive to CsA and that this differential effect of CsA is not related to the alloreactive potential of the infused T cells. These findings could be highly relevant when considering allogeneic T cell therapy approaches.

Chemoradiotherapy followed by surgery compared with surgery alone in squamous-cell cancer of the esophagus.

BACKGROUND: We conducted a multicenter, randomized trial to compare preoperative chemoradiotherapy followed by surgery with surgery alone in patients with stage I and II squamous-cell cancer of the esophagus. METHODS: The preoperative combined therapy consisted of two one-week courses; each involved radiotherapy, in a dose of 18.5 Gy delivered in five fractions of 3.7 Gy each, and 80 mg of cisplatin per square meter of body-surface area, administered 0 to 2 days before the first day of radiotherapy. The surgical plan included one-stage en bloc esophagectomy and proximal gastrectomy by the abdominal and right thoracic routes, to be performed immediately after randomization in the group assigned to surgery alone and two to four weeks after the completion of preoperative chemoradiotherapy in the group assigned to combined therapy. RESULTS: A total of 297 patients entered the study; 11 were found to be ineligible, and 4 were lost to follow-up. Of the remaining 282, 139 were assigned to surgery alone and 143 to combined therapy. After a median follow-up of 55.2 months, no significant difference in overall survival was observed; the median survival was 18.6 months for both groups. As compared with the group treated with surgery alone, the group treated preoperatively had longer disease-free survival (P=0.003), a longer interval free of local disease (P=0.01), a lower rate of cancer-related deaths (P=0.002), and a higher frequency of curative resection (P=0.017). However, there were more postoperative deaths (P=0.012) in the group treated preoperatively with chemoradiotherapy. Three prognostic factors were found to influence survival in a multivariate analysis: the disease stage, based on computed tomography; the location of the tumor; and whether the surgical resection was curative. CONCLUSIONS: In patients with squamous-cell esophageal cancer, preoperative chemoradiotherapy did not improve overall survival, but it did prolong disease-free survival and survival free of local disease.

[Radiotherapy and symptomatic treatments of advanced colorectal cancers]. / Radiothérapie et traitements symptomatiques des cancers colo-rectaux évolués.

Treatments of colorectal cancers can still be curative up to fairly advanced stage. Clinical situations vary greatly depending on the primary: local recurrence is a major event in rectal cancer whereas metastases are the main problem in colon cancer. Therefore, rectal cancer benefit from locoregional combined treatments, chemotherapy, radiotherapy and surgery, to control the pelvic disease. In the metastatic setting, it is important to offer potentially curative treatment as often as possible. Radiotherapy for bone or brain metastasis is well known and frequently used; irradiation of painful liver metastasis is less regularly discussed despite its efficacy. Symptomatic treatments like analgesics narcotic or not, non steroidal anti-inflammatory, anxiolytics, etc. are useful. All these treatments must be evaluated, not only in term of anti-tumoural but also from the quality of life point of view.

[Scoring system of late effects of radiations on normal tissues: the SOMA-LENT scale]. / Système d'évaluation des effets tardifs des radiations sur les tissus normaux: l'échelle SOMA-LENT. Groupe de travail des effets tardifs de l'EORTC.

Radiation tolerance of normal tissues remains the limiting factor for delivering tumoricidal dose. The late toxicity of normal tissues is the most critical element of an irradiation: somatic, functional and structural alterations occur during the actual treatment itself, but late effects manifest months to years after acute effects heal, and may progress with time. The optimal therapeutic ratio ultimately requires not only complete tumor clearance, but also minimal residual injury to surrounding vital normal tissues. The disparity between the intensity of acute and late effects and the inability to predict the eventual manifestations of late normal tissue injury has made radiation oncologists recognize the importance of careful patient follow-up. There is so far no uniform toxicity scoring system to compare several clinical studies in the absence of a "common toxicity language". This justifies the need to establish a precise evaluation system for the analysis of late effects of radiation on normal tissues. The SOMA/LENT scoring system results from an international collaboration. European Organization Treatment of Cancer (EORTC) and Radiation Therapy Oncology Group (RTOG) have created subcommittees with the aim of addressing the question of standardized toxic effects criteria. This effort appeared as a necessity to standardize and improve the data recording, to then describe and evaluate uniform toxicity at regular time intervals. The current proposed scale is not yet validated, and should be used cautiously.

[Late effects of radiations on the esophagus]. / Effets tardifs des radiations sur l'oesophage.

Radiation-induced late effects of oesophagus are observed after treatment for various cancers. Acute reactions, mainly oesophagitis, are well known and accurately described; late effects share, for most of these, a common consequence: alteration of the main oesophageal function, namely to conduct the food bolus; clinically they are impaired in terms of mobility and stenosis. More rarely, ulcerations and pseudodiverticulae can be observed. Chemotherapy further increases the risk of late effects, especially in case of concomitant chemo-radiotherapy. All numbers and statistical data on oesophagus late effects should be regarded with caution due to recent changes in the therapeutic attitudes (more and more combined chemotherapy-radiotherapy) and some progress in given cancer locations. A common scale like the LENT-SOMA should enable the clinician to better know these late effects on oesophagus which is required to initiate effective prevention measures and adapted treatments.

[Late intestinal complications of adjuvant radiotherapy of rectal cancers]. / Les complications intestinales tardives de la radiothérapie adjuvante des cancers rectaux.

Pre or postoperative pelvic irradiation has demonstrated a definitive efficacy in reducing the local failure rate of rectal cancer treated with surgery alone. However it can induce late small bowel morbidity that could alter the therapeutic ratio. The clinical pictures of radiation enteritis include obstruction and diarrhea/malabsorption. Prognostic factors that increase the risk of late small bowel complications include extended fields out of the pelvis, irradiation dose, inappropriate irradiation technique, and increased small bowel irradiated volumes. The addition of chemotherapy increases acute but not late toxicity. Recommendations concerning the clinical practice are described. Radiotherapy may also alter the residual sphincter function and we recommend to assess correctly these complications.

[Predictive tests of response to radiotherapy. Assessment and perspectives in 1997]. / Les tests prédictifs de la réponse à la radiothérapie: bilan et perspectives en 1997.

The potential tailoring of radiotherapy modalities to the biological characteristics of individual tumours and normal tissues appears to be an exciting way to improve the therapeutic, ratio in radiation therapy patients. Numerous assays have been proposed to provide the clinician with the biological information necessary to predict the outcome after irradiation and to guide the treatment prescription, but none of them has made its way to daily practice. Major difficulties are due to the technical burden of the procedures, the poor characterization of the assayed cells, and, moreover, the high complexity of tumour and normal tissues biology. The present paper reviews the present status of the assessment of tumour cells radiosensitivity, proliferation and oxygenation. Research remains extremely active in the field of biological predictors of response to irradiation. Future steps forwards are expected from progress in the available technologies, (re-)discovery of apoptosis and investigation of normal tissue tolerance.

Evidence that a transient enhancement of endogenous hematopoiesis contributes significantly to the favorable outcome following interleukin 1 pretreatment and allogeneic bone marrow transplantation.

The administration of IL-1, a potent radioprotective cytokine, before allogeneic BMT is associated with an early transient increase of circulating granulocytes, successful engraftment, and accelerated multilineage hematopoietic recovery. We have examined the effects of IL-1 alpha pretreatment on the engraftment of an allogeneic BMT unable to sustain survival by itself after a lethal irradiation: (1) transplantation of a limited amount of marrow cells and (2) transplantation several days after irradiation. IL-1 was unable to allow the engraftment of an early quantitatively inadequate BMT. However, delayed BMT with limited amounts of marrow cells was associated with engraftment in IL-1 pretreated recipients. Engraftment of a late (day 12) BMT in these IL-1-pretreated mice was comparable to the engraftment of a similar day 12 allogeneic BMT in non-IL-1-pretreated mice rescued from the lethal irradiation by an early (day 1) syngeneic graft. These findings demonstrate that IL-1 pretreatment can result in a dissociation between BMT-induced survival and engraftment and suggest that the favorable effects of IL-1 pretreatment in an allogeneic BMT setting are mainly mediated through a transient enhancement of endogenous hematopoiesis and not through a direct effect on the allogeneic stem cells present in the marrow graft.

[Concomitant preoperative radiochemotherapy for rectal cancers]. / Radio-chimiothérapie concomitante pré-opératoire des cancers rectaux.

In December 1994, The French Consensus Conference on the Treatment of Rectal Cancer stated that: "Preoperative irradiation improves local control in T3 and T4 resectable rectal cancer patients and is recommended". Recent data indicate that this approach not only reduces local failure but also increases overall survival. The therapeutic ratio depends on the total dose delivered and technical parameters. Inadequate radiotherapy techniques leeds to increased toxicity and masks the potential benefits of the treatment. Combined preoperative chemo-radiotherapy, a promising issue in rectal cancer patients, is now under evaluation in a large European phase III trial (EORTC 22921).