A split-organ delineation approach for dose optimisation for intensity-modulated radiotherapy for advanced T-stage nasopharyngeal carcinoma.

AIMS: To assess the dosimetric effect of using a split-organ delineation approach during intensity-modulated radiotherapy (IMRT) treatment planning for advanced T-stage nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: Twenty NPC patients with T3-4 tumours were studied. A reference (REF) IMRT plan was generated based on a standard treatment planning protocol, with a set of user-defined dose constraints for optimisation. An investigative (INV) IMRT plan was then generated based on the same protocol, but treating several organs at risk (OARs; parotid glands, temporal lobes, cochlea, auditory nerves and planning organ at risk volume [PRV] of the brainstem) as split organs consisting of target-overlapping and non-target-overlapping sub-segments. These sub-segments were assigned independent dose constraints. The REF and INV plans were compared with respect to target coverage and OAR sparing. Target coverage was evaluated by the Dmin (minimum dose), V66/V60 (percentage volume of gross target volume [GTV]/planning target volume [PTV] receiving 66 Gy/60 Gy), target conformity index (CI), and tumour control probability (TCP). The sparing of OARs was evaluated by the commonly used dose end points for the respective OAR, and normal tissue complication probability (NTCP). RESULTS: For PTV coverage, the INV plan was superior to the REF plan in terms of Dmin (P=0.000), CI (P=0.005) and TCP (P=0.002). This is attributed to an increase in dose to the PTV-OAR overlapping sub-segments. Regarding the sparing of OARs, there was a significant reduction in the mean dose of the parotid glands (P=0.002), and a slight, but non-significant, increase in NTCP of the temporal lobes, cochlea and brainstem. CONCLUSIONS: Using a split-organ delineation approach in IMRT treatment planning for advanced T-stage NPC, a significant improvement in the target coverage and TCP could be achieved, whereas the mean dose of the parotid was reduced significantly. There was insignificant change in the NTCP of the temporal lobe, parotid gland, cochlea and brainstem, but a significant change in the NTCP of the auditory nerve. The approach provides the planner extra room to manipulate the dose constraints during optimisation, and to obtain the desired result in less attempts. This approach also has the potential to be used in a broader context for IMRT planning for other tumour sites.

Three-dimensional dosimetric evaluation of a conventional radiotherapy technique for treatment of nasopharyngeal carcinoma.

BACKGROUND AND PURPOSE: The aim of this study is to evaluate and delineate the deficiencies in conventional two-dimensional (2-D) radiotherapy planning of nasopharyngeal carcinoma (NPC) treatment and to explore the means for improvement of the existing treatment technique aiming at enhancing local tumor control and reducing treatment complications. METHODS AND MATERIALS: Ten patients with NPC sparing the skull base and without intracranial extension or cranial nerve(s) palsy were chosen in the present study. Two sets of CT images for Phases I and II of the radiotherapy treatment were taken with patient immobilized in the flexed-head and the extended-head positions, respectively. Based on the CT images and endoscopic findings, the gross tumor volume (GTV) was defined. The clinical target volume (CTV) circumscribing the GTV was defined according to Ho's (Halnan, K.E. (ed.) Treatment of Cancer. London: Chapman and Hall, 1982. pp. 249-268) description of the organs at risk of tumor infiltration. The planning target volume (PTV) was defined by adding a margin to the CTV which catered for geometrical inaccuracies. The field borders and shields were set at standard distances from certain bony landmarks and were drawn on the simulator radiograph. Data on the beams and shield arrangements were then transferred to the planning computer via a digitizer. By applying 3-D volumetric dose calculation using a commercial three-dimensional (3D) treatment planning computer, the dose-volume-histograms (DVHs) of GTV, CTV, PTV and critical normal organs were generated for both phases of Ho's treatment technique. The same patients were re-planned using a modified Ho's technique which used 3-D beams-eye-view (BEV) in placing the shielding blocks and the same set of DVHs were generated and compared with those obtained from Ho's technique. RESULTS: The median volumes of GTV, CTV and PTV covered by the 95% isodose in Ho's phase I treatment were around 60%. The dose coverage was unsatisfactory in the superior and inferior and the posterolateral regions. In phase II treatment, the median volume of GTV, CTV and PTV covered by the 95% isodose were 99, 96 and 72%, respectively. Even though the dose coverage of the PTV in both phases of treatment were unsatisfactory, radiotherapy with the original Ho's technique had consistently produced good local control for NPC. However, there is potential room for enhancing the local control further because after modifying Ho's technique by using 3-D BEV customization of the treatment portals, the median volume of the target covered by the 95% isodose was defined as V(95). The V(95) of the PTV during the Phase II treatment was improved by 13%. The 90% of the volume of temporo-mandibular joints and parotid glands were both irradiated to 53 Gy and 43.6 Gy of the total prescribed dose of 66 Gy, respectively, in phase I and II treatments. With the addition of a hypothalamus-pituitary shield to Ho's technique, 50% of the volume of optic chiasma and temporal lobes received, respectively, 19.3 Gy and 4.5 Gy. However, small volume of the temporal lobes received a maximum dose (D(max)) of 62.8 Gy (95.2% of 66Gy). Most of the brainstem was shielded from the lateral portals but 5% of its volume received a dose ranging from 25.4 to 50.4Gy. The spinal cord (at C1/C2 level) received a D(max) of 40.8 Gy in phase I and of 4.8 Gy in phase II. After modifying Ho's technique by 3-D BEV customization of the treatment portals, the D(max) to the brainstem, the optic chiasma and the temporal lobes could be reduced by 8, 12 and 5%, respectively. CONCLUSIONS: Our study indicated that the dose-coverage of the PTV in Ho's radiotherapy technique for the early T-stage NPC was less than satisfactory in the superior and inferior and the posterolateral regions. However, in view of the excellent historical local tumor control with Ho's technique, we have to postulate that the present definition of CTV (and hence the PTV after adding margins to the CTV) lacks clinical significance and can be improved. It appears that the inclusion of the entire sphenoid sinus floor and both medial and lateral pterygoid muscles in the CTV is not necessary for maximal tumor control in the absence of clinical/radiological evidence of tumor infiltration of these organs. Ho's technique can be improved by using 3-D BEV to customize the treatment portals with multileaf collimators or blocks.

Final report of a randomized trial on altered-fractionated radiotherapy in nasopharyngeal carcinoma prematurely terminated by significant increase in neurologic complications.

PURPOSE: The aim of the present study was to compare the survival, local control and complications of conventional/accelerated-hyperfractionated radiotherapy and conventional radiotherapy in nonmetastatic nasopharyngeal carcinoma (NPC). METHODS AND MATERIALS: From February 1993 to October 1995, 159 patients with newly diagnosed nonmetastatic (M0) NPC with N0 or 4 cm or less N1 disease (Ho's N-stage classification, 1978) were randomized to receive either conventional radiotherapy (Arm I, n = 82) or conventional/accelerated-hyperfractionated radiotherapy (Arm II, n = 77). Stratification was according to the T stage. The biologic effective dose (10 Grays) to the primary and the upper cervical lymphatics were 75.0 and 73.1 for Arm I and 84.4 and 77.2 for Arm II, respectively. RESULTS: With comparable distribution among the T stages between the two arms, the free from local failure rate at 5 years after radiotherapy was not significantly different between the two arms (85.3%; 95% confidence interval, 77.2-93.4% for Arm I; and 88.9%; 95% confidence interval, 81.7-96.2% for Arm II). The two arms were also comparable in overall survival, relapse-free survival, and rates of distant metastasis and regional relapse. Conventional/accelerated-hyperfractionated radiotherapy was associated with significantly increased radiation-induced damage to the central nervous system (including temporal lobe, cranial nerves, optic nerve/chiasma, and brainstem/spinal cord) in Arm II. Although insignificant, radiation-induced cranial nerve(s) palsy (typically involving VIII-XII), trismus, neck soft tissue fibrosis, and hypopituiturism and hypothyroidism occurred more often in Arm II. In addition, the complications occurred at significantly shorter intervals after radiotherapy in Arm II. CONCLUSION: Accelerated hyperfractionation when used in conjunction with a two-dimensional radiotherapy planning technique, in this case the Ho's technique, resulted in increased radiation damage to the central nervous system without significant improvement in efficacy.

The roles of multileaf collimators and micro-multileaf collimators in conformal and conventional nasopharyngeal carcinoma radiotherapy treatments.

The purpose of this work is to study the efficacy and limitations of using standard multileaf collimators (MLCs) and micro-multileaf collimators (mMLCs) in the treatment of nasopharyngeal carcinoma (NPC) by conventional and conformal radiotherapy techniques. The penumbra characteristics of MLC, mMLC, and customized block collimated beams are measured with respect to leaf edge angle, beam energy, treatment depth, and field size and compared with those generated by a commercial three-dimensional planning computer system. Upon verification of the planning system, it is used to evaluate the treatment plans generated with these beam shapers for conventional and conformal NPC treatments. The effective penumbra of a MLC beam is strongly influenced by its edge angle, leaf width, and treatment depth. The suitability of standard MLCs in conventional NPC treatments is determined mainly by the edge angle to be used. For conformal NPC treatments involving six or more fields, dose volume histograms comparable to those of customized beam blocks are obtained with a standard MLC. The mMLC does not have the same restrictions as those on standard MLC but is limited to phase II treatment by its small usable field size. Both standard MLCs and mMLCs can be used to replace customized divergent beam blocks in both conventional and conformal NPC treatments. However, a MLC, due to its larger effective penumbra, may be unsuitable for use in cases when the tumor volumes extend very close to the critical normal structures. A mMLC, on the other hand, is limited by its small maximum field size and can only be used for collimating the facial portals in the second phase treatment.

Increased incidence of tongue cancer after primary radiotherapy for nasopharyngeal carcinoma--the possibility of radiation carcinogenesis.

The aim of this study was to define the risk of tongue and other aerodigestive tract cancers developing after primary radiation therapy for nasopharyngeal carcinoma (NPC). A cohort of 903 patients with non-disseminated NPC given radical radiotherapy between 1984 and 1989 was studied for the incidence of tongue cancer and other malignancies during follow-up. A contemporary cohort of 87 patients with tongue cancer, without a history of NPC, was studied for demographic data, cigarette smoking and alcohol consumption habits. These were then compared with all the NPC patients and with the NPC patients who later developed tongue cancers. There was a significantly increased number of tongue cancers following radiotherapy for NPC. The risk of developing tongue cancer after radiotherapy for NPC was 0.13% per patient per year. There was no increase in the number of other malignancies. The association between NPC and tongue cancer was that of a non-random temporal sequence with tongue cancers following NPC but not in the reverse order. The demographic data and smoking and alcohol consumption history of the 7 NPC patients who subsequently developed tongue cancer were significantly different from the de novo tongue cancer patient population. The absence of common aetiological factors between NPC and tongue cancer and the non-random sequence of tongue cancers occurring after NPC suggests that these seven tongue cancers could be radiation induced. The estimated radiation dose received by the part of the tongue developing cancer was substantial and significantly higher than the dose to the cancer-free tongue. An increase of tongue cancers after radiotherapy for NPC is reported and arguments are made in support of the hypothesis that these were radiation-induced malignancies. We suggest a decrease in the volume of tongue included within the planning target volume of NPC in the absence of oropharyngeal and/or parapharyngeal infiltration. Awareness of the association should make early diagnosis of this likely radiation-induced cancer possible.

[Cell cycle and apoptosis].

The progression of cell cycle in embryonic cells from oceanic bionts to mammalians is initiated, promoted and terminated under the regulation of cell cycle gene products, named cyclins, and a p34 (cdc 2). Besides, oncogene (proto-oncogene) products such as p53 and pRB also directly regulate the progression of cell cycle. However, the p34 (cdc 2) which promotes the mitotic cell division also initiates the apoptosis of certain cells. Therefore, mutations of genes that regulate the normal progression of cell cycle would cause cells in the cell cycle undergoing either apoptosis or uncontrollable proliferation.

[Anti-retinoblastoma reaction developed in vivo through immunization of retinal ganglion neuronotrophic factor].

Isolated retinal ganglion neuronotrophic factor (RGNTF) was used as an antigen to immunize Group A Balb/c mice intraperitoneally prior to the inoculation into the anterior chamber of eye with human retinoblastomal cell line Y-79 (Rb). In Group B mice, Rb cells were inoculated into the eyes before RGNTF immunization. In Group C mice, empty gel without RGNTF was used in immunization 10 days after the Rb inoculation, to serve as a control. The results revealed that the inhibitory rate of Rb tumor development in Group A was 65% (13/20); in Group B only 10% (2/20); and in Group C 0% (0/20). The T-test for difference in the inhibitory rate between Group A and B was statistically significant (T > 2.58; P < 0.01). Sera were collected from these mice and their content of the anti-RGNTF antibody was quantified by ELISA method. The results showed that the anti-RGNTF antibody titer in Group A antisera at 1:600 dilution was measured with an average optical density of 0.156 +/- 0.015; that in Group B 0.103 +/- 0.016; and that in Group C only 0.048 +/- 0.018. Those of controls for normal mouse serum and culture medium were 0.050 +/- 0.008 and 0.043 +/- 0.014, respectively. The t test for difference in the antibody titer measurements between Group A and B was statistically significant (t > t0.05; p < 0.05). Therefore, the above results indicated that active immunization of RGNTF can enhance the specific immunity against the development of retinoblastomal tumor in Balb/c mice, which may have clinical significance in treating human retinoblastoma.

Development of choline acetyltransferase-immunoreactive neurons in normal and intracranially transplanted retinas in rats.

Retinas from embryonic day 14 (E14) Sprague-Dawley rats were transplanted to the tectum of newborn (P0) recipient rats, and the distribution pattern of choline acetyltransferase immunoreactivity (ChAT-I) in developing transplants was studied and compared with those observed in the retinas of normal developing rats. In normal retinas, ChAT-I cells were first identified in restricted regions in the ganglion cell layer (GCL) at P4, but were found to cover the entire GCL by P6. A second population of ChAT-I cells was detected in the inner nuclear layer (INL) at P8, and they were observed in most parts of the INL on P10 when two immunoreactive sublaminae began to appear in the inner plexiform layer (IPL). The adult pattern of having two distinct populations of ChAT-I cells, organized in mirror symmetrical fashion in the inner retinal layers was basically established by P12. The time course of development and overall distribution pattern of ChAT-I cells in developing retinal transplants on the whole were very similar to those observed in normal retinas. The first identification of these cells and the establishment of their final distribution pattern were made at stages corresponding to P4 and P12 of normal developing retinas respectively. However, ChAT-I somata were located in the INL at a much earlier stage compared with their counterparts in the normal retina, and a transient population of immunoreactive cells with their processes extending to retinal layers other than the IPL was observed in some transplants from P6 to P10. These features were not observed in normal developing retinas. These results suggest that the development of cholinergic neurons, especially the expression of their characteristic antigen and their final distribution pattern is largely determined by programmes which are intrinsic to the original retinal tissue, despite some minor deviation or variation in the developmental process which may occur under certain abnormal conditions.

Induction of helper T cells by antigen specific factor from macrophages.

An immunostimulatory antigen specific factor (ASF) was found to be secreted by antigen-pulsed macrophages. Macrophages obtained from peritoneal exudate of C57BL/6 mice were pulsed with horse spleen ferritin (HSF). The PM10 ultrafiltration membrane-retained supernatant from the cultures of these macrophages was able to generate helper T cells when introduced into cultures of nylon wool purified T lymphocytes from spleens of C57BL/6 mice. The generation of helper T cells was measured by the cooperation between ASF-induced T cells and splenic B cells in the presence of trinitrophenyl (TNP)-HSF, and subsequently by the anti-TNP plaque forming cell (PFC) assay using TNP-coated sheep red blood cells. The number of PFC obtained from these cultures was significantly higher than the control (T cell cultures without ASF). Background levels of PFC were obtained when the T-B cooperation cultures were challenged with other haptenated antigens (e.g. TNP-BSA) instead of TNP-HSF. In addition, ASF from allogeneic macrophages was unable to facilitate helper cell induction. These results indicated that helper T cell induction by ASF is antigen specific as well as genetically restricted. When small amounts of ASF were injected into syngeneic mice without any adjuvant, specific helper T cells could be obtained from the spleens of these animals which showed that ASF is also active in vivo.

The cholinergic system in retinal transplants in rats.

Retinas from embryonic day 13 or 14 Sprague-Dawley albino rats were transplanted to the brainstem of newborn rats with unilateral eye enucleation at birth. Two months after the transplantation, the activity and distribution of acetylcholinesterase and choline acetyltransferase were studied using histochemical and immunocytochemical methods respectively. Results obtained showed that the staining patterns of these two cholinergic enzymes in the retinal transplants were essentially the same as those observed in the retinas of normal rats and in the control retinas of the recipient animals. The similarities in the distribution of these two cholinergic enzymes in these retinas suggest that the cholinergic system in the retinal transplants is likely to be functional.

Factors affecting neurite outgrowth of occipital cortical explants.

The effects of various substrata including laminin, collagen gel, collagen I, and human amniotic basement membrane on neurite outgrowth of occipital cortical and diencephalic explants were studied. The results showed that the extent and pattern of growing neurites of cortical explants varied considerably depending on the substrata used. While an elaborated network of growing neurites was observed when cortical explants were plated on laminin, the most extensive neurite outgrowth was observed when collagen gel was used as the substratum. In contrast, diencephalic explants did not grow on most of the substrata. The significance of the findings are discussed.

Preservation of the entire population of normally transient ipsilaterally projecting retinal ganglion cells by neonatal lesions in the rat.

The number of ipsilaterally projecting retinal ganglion cells (IPRGCs) in developing normal rats and rats which received unilateral thalamic lesion and monocular enucleation at birth was studied using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) as a retrograde neuronal marker. The results showed that the number of IPRGCs labelled in day-21 rats which received lesions at birth was comparable to the highest number of IPRGCs observed in normal rats on day 0 (day of birth). These results suggest that the entire population of IPRGCs which had their axons already grown in or near to their target structures on day 0 can be rescued by neonatal lesions.

Cytochrome oxidase activity in retinas transplanted to the brainstem in rats.

Fetal retinas were transplanted to the brainstem of newborn rats and their morphological features were examined using the cytochrome oxidase histochemical method at maturity. The results showed that the inner segments of photoreceptors, outer and inner plexiform layers as well as ganglion cells with large somata were moderately to darkly stained for cytochrome oxidase. This pattern is basically the same as that observed in the normal retina, suggesting that cytochrome oxidase can be used not only for revealing spatial but also functional organization of retinal transplants.

Cytochrome oxidase activity in the rat retina following unilateral thalamic lesion.

The present study has investigated the cytochrome oxidase (CO) activity in retinas of normal rats and rats which received central lesions at birth or young adult stage. The results show that a thalamic lesion which injured the retinal ganglion cell axons in young adult rats led to severe loss of CO activity in the ganglion, inner and outer plexiform layers in the retina contralateral to the lesion as compared to those of normal rats. In contrast, distinct CO-reactive bands and cells were clearly observed in corresponding laminae in retinas in which almost the entire population of retinal ganglion cells was eliminated by a neonatal thalamic lesion. These results indicate that retinal ganglion cells contribute significantly to the CO activity observed in the inner retinal laminae under normal but not under abnormal conditions, and suggest that considerable changes in the activity of the remaining neurons and possibly reorganization of neural circuitry within the retina in rats which received neonatal lesions has taken place, as revealed by CO histochemistry.

Peroxidative block of glucose utilization and survival in CNS neuronal cultures.

The search for neuronotrophic factors addressing CNS neurons requires CNS neuronal cell cultures to quantitate putative effects on neuronal survival. Investigation of neurons dissociated from several embryonic CNS tissues have shown that their short-term survival requires supplementation of the culture medium with either pyruvate or the enzyme catalase. Pyruvate can be replaced with alpha-ketoglutarate or oxaloacetate, or with amino acids capable to transaminate to these three metabolites in the presence of exogenous alpha-ketoacid acceptors. Experiments were designed to evaluate the ability of cultured CNS neurons to utilize glucose as their primary source. We show that: (1) catalase requires the availability of glucose in the medium in order to exert its neuronal maintenance effect, (2) in the absence of catalase, the cells are unable to metabolize glucose through the tricarboxylic acid cycle, (3) catalase restores the neuronal ability to utilize glucose for oxydative metabolism, and renders redundant the use of other sources such as glutamate conversion to alpha-ketoglutarate, (4) graded concentrations of glucose in the medium affect in parallel these metabolic activities and the viability of the cultured neurons, and (5) anti-oxidant agents other than catalase mimic the catalase effects. We conclude that dissociated embryonic CNS neurons suffer from a block in glucose utilization which results from an imbalance between free radical attack and cellular defenses to it and speculate on a more general involvement of peroxidation damage in the trophic requirements for neuronal survival.

Calorimetric and freeze fracture analysis of lipid phase transitions and lateral translational motion of intramembrane particles in mitochondrial membranes.

Differential scanning calorimetry combined with freeze fracture electron microscopy reveals that thermotropic lipid phase transitions and lateral translational motion of intramembrane particles occur in both membranes of whole, intact rat liver mitochondria and in isolated inner and outer membranes. The onset temperature of the liquid crystalline to gel state lipid phase transition in whole mitochondria and in the isolated outer membrane fraction is biphasic with an initial transition exotherm occurring at 9 degrees C. The onset temperature of the transition exotherm of the isolated inner membrane occurs at -4 degrees C. The onset temperature of the lipid transition endotherm is -15 degrees C for whole mitochondria, the inner membrane, ane the outer membrane fractions. These calorimetric analyses reveal that the bilayer lipid in the inner, energy transducing membrane is more fluid than in the outer membrane. Mitochondrial membranes cooled to temperatures in the region of their transition exotherms and then frozen reveal striking lateral separations between smooth, intramembrane particle-free regions (rich in gel state lipid) and particle-dense regions (rich in integral proteins) in their hydrophobic fracture faces. Such thermotropic lipid-protein lateral separations are completely reversible. These freeze fracture observations suggest that both mitochondrial membranes are naturally fluid to the extent that the integrat membrane proteins can diffuse laterally in the bilayer lipid.