Automatic segmentation of thoracic and pelvic CT images for radiotherapy planning using implicit anatomic knowledge and organ-specific segmentation strategies.

Automatic segmentation of anatomical structures in medical images is a valuable tool for efficient computer-aided radiotherapy and surgery planning and an enabling technology for dynamic adaptive radiotherapy. This paper presents the design, algorithms and validation of new software for the automatic segmentation of CT images used for radiotherapy treatment planning. A coarse to fine approach is followed that consists of presegmentation, anatomic orientation and structure segmentation. No user input or a priori information about the image content is required. In presegmentation, the body outline, the bones and lung equivalent tissue are detected. Anatomic orientation recognizes the patient's position, orientation and gender and creates an elastic mapping of the slice positions to a reference scale. Structure segmentation is divided into localization, outlining and refinement, performed by procedures with implicit anatomic knowledge using standard image processing operations. The presented version of algorithms automatically segments the body outline and bones in any gender and patient position, the prostate, bladder and femoral heads for male pelvis in supine position, and the spinal canal, lungs, heart and trachea in supine position. The software was developed and tested on a collection of over 600 clinical radiotherapy planning CT stacks. In a qualitative validation on this test collection, anatomic orientation correctly detected gender, patient position and body region in 98% of the cases, a correct mapping was produced for 89% of thorax and 94% of pelvis cases. The average processing time for the entire segmentation of a CT stack was less than 1 min on a standard personal computer. Two independent retrospective studies were carried out for clinical validation. Study I was performed on 66 cases (30 pelvis, 36 thorax) with dosimetrists, study II on 52 cases (39 pelvis, 13 thorax) with radio-oncologists as experts. The experts rated the automatically produced structures on the scale 1-excellent (no corrections necessary, maximum time saving), 2-good (corrections necessary for up to 1/3 of slices), 3-acceptable (major corrections necessary, but still time saving), 4-not acceptable (manual redrawing more efficient, no time saving). A rating

Radiosensitivity in breast cancer assessed by the Comet and micronucleus assays.

Spontaneous and radiation-induced genetic instability of peripheral blood mononuclear cells derived from unselected breast cancer (BC) patients (n=50) was examined using the single-cell gel electrophoresis (Comet) assay and a modified G2 micronucleus (MN) test. Cells from apparently healthy donors (n=16) and from cancer patients (n=9) with an adverse early skin reaction to radiotherapy (RT) served as references. Nonirradiated cells from the three tested groups exhibited similar baseline levels of DNA fragmentation assessed by the Comet assay. Likewise, the Comet analysis of in vitro irradiated (5 Gy) cells did not reveal any significant differences among the three groups with respect to the initial and residual DNA fragmentation, as well as the DNA repair kinetics. The G2 MN test showed that cells from cancer patients with an adverse skin reaction to RT displayed increased frequencies of both spontaneous and radiation-induced MN compared to healthy control or the group of unselected BC patients. Two patients from the latter group developed an increased early skin reaction to RT, which was associated with an increased initial DNA fragmentation in vitro only in one of them. Cells from the other BC patient exhibited a striking slope in the dose-response curve detected by the G2 MN test. We also found that previous RT strongly increased both spontaneous and in vitro radiation-induced MN levels, and to a lesser extent, the radiation-induced DNA damage assessed by the Comet assay. These data suggest that clinical radiation may provoke genetic instability and/or induce persistent DNA damage in normal cells of cancer patients, thus leading to increased levels of MN induction and DNA fragmentation after irradiation in vitro. Therefore, care has to be taken when blood samples collected postradiotherapeutically are used to assess the radiosensitivity of cancer patients.

Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients.

About 5% of oncology patients treated by radiation therapy develop acute or late radiotoxic effects whose molecular mechanisms remain poorly understood. In this study, we evaluated the potential role of DNA repair proteins in the hypersensitivity of cancer patients to radiation therapy. The expression levels and focal nuclear distribution of DNA repair proteins, hMre11, Rad50 and Rad51 were investigated in skin fibroblasts strains derived from cancer patients with adverse early skin reaction to radiotherapy using Western blot and foci immunofluorescence techniques, respectively. Cells from cancer patients with normal reaction to radiotherapy as well as cells from apparently healthy subjects served as controls. Cellular radiosensitivity after in vitro irradiation was assessed by the clonogenic survival assay. The clonogenic survival assay and Western blot analysis of the DNA repair proteins did not reveal any abnormalities in cellular radiosensitivity in vitro and in protein expression levels or their migration patterns in the fibroblasts derived from cancer patients with hypersensitive reaction to radiotherapy. In contrast, in vitro irradiated cells from radiosensitive patients exhibited a significantly higher number of nuclei with focally concentrated Rad50 protein than in both control groups. The observed alteration of the distribution of radiation-induced Rad50 foci in cells derived from cancer patients with acute side reactions to radiotherapy might contribute to their radiation therapy outcome. These data suggest the usefulness of the Rad50 foci analysis for predicting clinical response of cancer patients to radiotherapy.

In vitro radiosensitivity measured in lymphocytes and fibroblasts by colony formation and comet assay: comparison with clinical acute reactions to radiotherapy in breast cancer patients.

PURPOSE: To compare colony-forming and comet assays on fibroblasts and lymphocytes of 32 breast cancer patients irradiated after breast-conserving operations and to correlate the results with acute clinical radiation reactions in the skin. MATERIAL AND METHODS: Skin fibroblasts were isolated and cultivated before radiotherapy and lymphocytes were drawn prior to the first and directly after the final external irradiation. The colony-forming assay was performed with fibroblasts and the comet assay with lymphocytes and fibroblasts of breast cancer patients according to standard protocols. The clinical radiation reactions of the patients were graded according to the RTOG system. RESULTS: No significant correlation (p =0.09) was detected between clinical acute skin reactions and the in vitro clonogenic data in fibroblasts. Results of the comet assay in lymphocytes, however, showed a significant correlation (p <0.05) with the clinical data when patients were divided into two groups with average and elevated acute reactions. Apart from initial damage, fibroblasts did not show significant differences between the two patient groups. Repeated comet assays in lymphocytes of the same patient drawn before treatment and before and after external radiotherapy demonstrated good reproducibility of the test and no significant impact of preceding radiation treatment. There was a good correlation (r =0.65) between the comet assay results in fibroblasts and lymphocytes of the same individual. CONCLUSIONS: In this cohort of patients, a significant correlation between the in vitro results of the comet assay in lymphocytes and clinical acute reactions was detected. The results of the comet assay and of fibroblast colony formation did not correlate with in vitro radiosensitivity.

Response to X-irradiation of Fanconi anemia homozygous and heterozygous cells assessed by the single-cell gel electrophoresis (comet) assay.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by bone marrow failure and cancer susceptibility. Patient cells are sensitive to a variety of clastogens, most prominently cross-linking agents. Although there is the long-standing clinical impression of radiosensitivity, in vitro studies have yielded conflicting results. We exposed peripheral blood mononuclear cells from FA patients and carriers to x-rays and determined their DNA damage and repair profiles using the alkaline single-cell gel electrophoresis (comet) assay. Studies were carried out in two independent series of experiments by two laboratories using different protocols. The cells of both FA patients and carriers showed uniformly high initial DNA damage rates as assessed by the total initial tail moment. In addition, the average residual tail moment at 30 to 50 minutes and the repair half-time parameters were significantly elevated. These findings suggest an increased release of fragmented DNA following x-ray exposure in cells that carry one or two mutations in one of the FA genes. The comet assay may be a useful adjunct for heterozygote detection in families of FA patients.

The in vitro colony assay: a predictor of clinical outcome.

PURPOSE: To determine the predictive power of an in vitro colony assay on the clinical normal-tissue complication rate. MATERIAL AND METHODS: Primary skin fibroblasts from 88 individuals were generated from the skin biopsies of patients who received a standardized radiotherapy. Tissue was cultured for three to six passages, irradiated with doses between 1 and 8 Gy under defined conditions, seeded and finally the colonies were stained and counted after 10-14 days. The survival curves were fitted by the L-Q model and the SF2, alpha/beta and plating efficiency were calculated. RESULTS: The parameters SF2 and plating efficiency were stable throughout the 4-year test period. Intra-individual differences between repeated experiments were significantly lower than inter-individual test results. For the observed acute skin and late normal-tissue reactions other than skin the in vitro parameter SF2 correlated significantly (p<0.005). For late skin reactions this correlation was not found. DISCUSSION: In contrast to other publications, a clear correlation was found between the in vitro test results and clinically observed early reactions. The lack of correlation for late skin reactions suggests that the combination of intrinsic radiation sensitivity and exogenous factors may alter the clinically observed reaction of certain tissues to a different extent.

Stereotactic radiotherapy of targets in the lung and liver.

BACKGROUND: Stereotactic irradiation of extracranial targets offers a non-invasive treatment modality for patients with localized tumors, which are not amenable for surgery or other invasive approaches because of age or impaired medical condition. The purpose of the study was the evaluation of the method to achieve local control of irradiated targets in relation to treatment toxicity. PATIENTS AND METHODS: Irradiation was performed as hypofractionated treatment in three fractions of 10 Gy each, normalized to the PTV enclosing 65% isodose with patient fixation in a stereotactic body frame. The isocenter was localized by stereotactic coordinates. Targets were circumscribed tumors in the lung (n = 27) and liver (n = 24) not amenable for other treatment modalities: primary lung cancer (n = 12), local recurrences of lung cancer (n = 4), lung metastases (n = 11), liver metastases (n = 23) and one cholangiocellular carcinoma. Median CTV/PTV for targets in the lung was 57/113 cm3 (min/max 5-277 cm3/17-343 cm3) and for targets in the liver 50/102 cm3 (min/max 9-516 cm3/42-772 cm3). Median follow-up for targets in the lung was 8 months (2-33) and 9 months (2-28) for liver targets. Local control was defined as complete or partial remission and stable disease, measured by repeated CT scans after 6 weeks and in 3 months intervals. Treatment toxicity was evaluated according to the WHO score. RESULTS: Crude local control was 85% for pulmonary targets and 83% for hepatic targets. Actuarial local control after 1 and 2 years was 76% and 76% for lung tumors and 76% and 61% for liver tumors. Actuarial overall patient survival was 48% after 1 year and 21% after 2 years for targets in the lung and 71% and 43% for targets in the liver. No acute grade 3-5 side effects were observed. Serious late toxicity occurred in two patients: a chronic ulceration of the esophagus at a target close to the mediastinum after 3 months (grade 3) and fatal bleeding from the pulmonary artery after 9 months (grade 5) in a previously irradiated patient. It remained unclear, whether the bleeding was a side effect of irradiation or due to tumor infiltration. CONCLUSION: Hypofractionated stereotactic irradiation of targets in the lung and liver is a locally effective treatment with actuarial local control rates of 76% after 1 year and 61-76% after 2 years without relevant acute toxicity. Severe late toxicity did not occur, if targets close to the mediastinum were avoided.

Stereotactic radiotherapy of extracranial targets: CT-simulation and accuracy of treatment in the stereotactic body frame.

BACKGROUND AND PURPOSE: Evaluation of set-up accuracy and analysis of target reproducibility in the stereotactic body frame (SBF), designed by Blomgren and Lax from Karolinska Hospital, Stockholm. Different types of targets were analyzed for the risk of target deviation. The correlation of target deviation to bony structures was analyzed to evaluate the value of bones as reference structures for isocenter verification. MATERIALS AND METHODS: Thirty patients with 32 targets were treated in the SBF for primary or metastatic peripheral lung cancer, liver metastases, abdominal and pelvic tumor recurrences or bone metastases. Set-up accuracy and target mobility were evaluated by CT-simulation and port films. The contours of the target at isocenter level, bony structures and body outline were compared by matching the CT-slices for treatment planning and simulation using the stereotactic coordinates of the SBF as external reference system. The matching procedure was performed by using a 3D treatment planning program. RESULTS: Set-up accuracy represented by bony structures revealed standard deviations (SD) of 3.5 mm in longitudinal, 2.2 mm in anterior-posterior and 3.9 mm in lateral directions. Target reproducibility showed a SD of 4.4 mm in longitudinal, 3.4 mm ap and 3.3 mm in lateral direction prior to correction. Correlation of target deviation to bones ranged from 33% (soft tissue targets) to 100% (bones). CONCLUSION: A security margin of 5 mm for PTV definition is sufficient, if CT simulation is performed prior to each treatment to correct larger target deviations or set-up errors. Isocenter verification relative to bony structures is only safe for bony targets but not for soft tissue targets.

The postoperative adjuvant radiation therapy and radiochemotherapy for UICC stage II and III rectal cancer. A retrospective analysis.

AIM: This analysis was undertaken to review the outcome and toxicity of postoperative adjuvant therapy for Stage II and III rectal cancer. PATIENTS AND METHODS: We reviewed 112 patients treated with radiotherapy (44 patients) and radiochemotherapy (68 patients) after potentially curative (R0) surgery for rectal cancer (UICC Stages II and III), between 1983 and 1994 at the University Clinic of Würzburg. Median radiation dose was 56 Gy (range: 45 to 66 Gy). Chemotherapy consisted of 4 to 6 courses of 5-fluorouracil (5-FU) (420 mg/m2/d) and leucovorin (200 mg/m2/d). Median follow-up was 37 months. RESULTS: The overall survival was 84% for patients with UICC Stage II and 45% for patients with UICC Stage III disease (p = 0.0045). There were no statistically significant differences between patients treated with radiochemotherapy vs radiotherapy in terms of 5-year survival (63% after radiochemotherapy vs 53% after radiotherapy, p = 0.16), relapse-free survival (52% vs 50%) and locoregional control (69% vs 67%). UICC Stage III disease was associated with high failure rates (40% pelvic recurrences and 53% distant metastases). There was a statistically significant difference in terms of the incidence of distant metastases between the 2 treatment modalities for patients with Stage III disease (49% 5-year probability for developing distant metastases after radiochemotherapy vs 66% after radiotherapy, p = 0.047). In a multivariate analysis, the addition of chemotherapy, lymph node stage and grading were independent prognostic factors for survival. Severe late toxicity was documented in 5% of treated patients. CONCLUSIONS: Prognosis of patients with UICC Stage III rectal cancer remains poor after "standard" surgery followed by postoperative adjuvant treatment (pelvic radiotherapy and bolus intravenous injection of 5-FU and leucovorin). Major efforts should be made in order to improve prognosis for these patients, including optimization of surgical treatment and systemic treatment. More effective multimodality treatment strategies should be investigated in prospective randomized trials.

Radiation-induced comet-formation in human skin fibroblasts from radiotherapy patients with different normal tissue reactions.

BACKGROUND: In clinical radiotherapy most patients tolerate the applied dosage with no or moderate side effects. However, 5 to 10% of all individuals show increased acute and/or late reactions. In-vitro test systems are investigated for their suitability for predictive purposes. This paper attempts a correlation between the induction and repair of DNA damage measured in the comet assay and the clinical observed reaction in order to evaluate the suitability of the comet assay for prediction of radiation sensitivity. PATIENTS AND METHODS: Skin fibroblasts of 30 patients with average tissue reactions or acute and/or late increased side effects and cell lines of 4 individuals carrying the heritable disease ataxia telangiectasia (AT) were irradiated in vitro. The induction and repair of DNA damage was measured at different time points after irradiation in the comet assay (single cell gel electrophoresis). These results were compared to the acute and late clinical reactions classified according to the RTOG grading system. RESULTS: The radiation induced DNA damage decreased over time reflecting DNA repair. Cells of the AT individuals showed an elevated damage induction and a reduced repair capacity compared to patients with average tissue reactions. Fibroblasts of patients with increased acute and late side effects exhibited slower DNA repair. In addition to the known lack of cell cycle control, our results indicate that AT cells show reduced DNA repair capacity. CONCLUSIONS: The comet assay seems to be able to detect some types of increased individual radiation sensitivity. In contrast to other predictive in-vitro tests, the comet assay needs less time and fewer cells, which would be useful in a clinical setting.

Sequence analysis of the ATM gene in 20 patients with RTOG grade 3 or 4 acute and/or late tissue radiation side effects.

PURPOSE: Patients with ataxia-telangiectasia (A-T) show greatly increased radiation sensitivity and cancer predisposition. Family studies imply that the otherwise clinically silent heterozygotes of this autosomal recessive disease run a 3.5 to 3.8 higher risk of developing cancer. In vitro studies suggest moderately increased cellular radiation sensitivity of A-T carriers. They may also show elevated clinical radiosensitivity. We retrospectively examined patients who presented with severe adverse reactions during or after standard radiation treatment for mutations in the gene responsible for A-T, ATM, considering a potential means of future identification of radiosensitive individuals prospectively to adjust dosage schedules. MATERIAL AND METHODS: We selected 20 cancer patients (breast, 11; rectum, 2; ENT, 2; bladder, 1; prostate, 1; anus, 1; astrocytoma, 1; Hodgkins lymphoma, 1) with Grade 3 to 4 (RTOG) acute and/or late tissue radiation side effects by reaction severity. DNA from the peripheral blood of patients was isolated. All 66 exons and adjacent intron regions of the ATM gene were PCR-amplified and examined for mutations by a combination of agarose gel electrophoresis, single-stranded conformational polymorphism (SSCP) analysis, and exon-scanning direct sequencing. RESULTS: Only 2 of the patients revealed altogether four heteroallelic sequence variants. The latter included two single-base deletions in different introns, a single-base change causing an amino acid substitution in an exon, and a large insertion in another intron. Both the single-base deletions and the single-base change represent known polymorphisms. The large insertion was an Alu repeat, shown not to give rise to altered gene product. CONCLUSIONS: Despite high technical efforts, no unequivocal ATM mutation was detected. Nevertheless, extension of similar studies to larger and differently composed cohorts of patients suffering severe adverse effects of radiotherapy, and application of new technologies for mutation detection may be worthwhile to assess the definite prevalence of significant ATM mutations within the group of radiotherapy patients with adverse reactions. To date, it must be recognized that our present results do not suggest that heterozygous ATM mutations are involved in clinically observed radiosensitivity but, rather, invoke different genetic predisposition or so far unknown exogenous factors.

Identification of ataxia telangiectasia heterozygotes, a cancer-prone population, using the single-cell gel electrophoresis (Comet) assay.

Heterozygotes of ataxia telangiectasia (AT) may comprise up to 1% of the general population. Because these individuals have no clinical expression of AT but may be highly radiosensitive and strongly predisposed for several forms of cancer, identification of AT carriers represents a considerable interest in cancer epidemiology and radiotherapy. We report a new approach for the in vitro identification of AT-heterozygotes based on the evaluation of the radiosensitivity and DNA damage repair ability of peripheral blood mononuclear cells using the single-cell gel electrophoresis (Comet) assay. The assay was performed on cells isolated from four different groups of individuals: (1) apparently healthy donors (n = 10); (2) patients with breast cancer showing a normal reaction to radiotherapy (n = 10); (3) a group of obligate AT carriers (parents of AT-homozygotes, n = 20); and (4) AT-homozygotes (n = 4). Cells irradiated with 3 Gy of x-rays were assayed for three parameters: (1) the initial and (2) residual DNA damage and (3) the kinetics of DNA damage repair. Both AT-heterozygotes' and AT-homozygotes' cells were found to be highly sensitive to x-irradiation. Quantitative evaluation of the single-cell electrophoregrams revealed that the average initial DNA damage in AT-heterozygous and AT-homozygous cells was almost three times higher than that in control non-AT cells. In addition, the DNA repair process in irradiated AT carrier cells was almost three times slower, and the extent of irreparable DNA damage in these cells was three times greater than in controls. Simultaneous assessment of the three parameters enabled correct identification of all tested AT carriers. This method seems to be a sensitive and useful tool for populational studies as a rapid prescreening test for a mutated AT status. The approach can also be extended for prediction of the in vivo radiosensitivity, which would enable optimization of individual radiotherapy schedules.

3D-recurrence-patterns of glioblastomas after CT-planned postoperative irradiation.

BACKGROUND AND PURPOSE: The introduction of computed-tomography as an advanced planning tool for the irradiation of intracranial tumours led to a controversial discussions about the optimal target-volume for the primary and postoperative treatment of malignant gliomas. This study analyses the three-dimensional tumour regrowth pattern relative to the treated volume which included the macroscopic preoperative tumour and 2-cm safety margin. MATERIALS AND METHODS: Seventy-nine patients with histologically-confirmed Glioblastoma multiforma and documented recurrence who were irradiated in our department between 1990 and 1996 were reviewed. With the help of a computer program written for this purpose, the PTV of the CT-based treatment plan was reconstructed and its spatial outline compared with the reconstructed volume of the recurrent tumour in the control CT-study. RESULTS: In 33 out 34 patients for which the CT-study showing tumour-recurrence was available the recurrence was completely situated within the original 90%-isodose. Only one tumour surpassed the outside surface of the PTV but was predominantly situated within the original tumourbed and suggests a tumour-regrowth within the high dose volume. CONCLUSIONS: The above results show that target-volumes based on the preoperative size of the enhanced tumour mass well cover the site of recurrence in nearly all cases. The findings suggest dose escalation to a more restricted volume.

Radiation-induced micronucleus formation in human skin fibroblasts of patients showing severe and normal tissue damage after radiotherapy.

PURPOSE: Treatment schedule and total dosage in radiation therapy is based on the tumoricidal doses and the tolerance dose of the perifocal normal tissue. Since large-scale variations occur between the patients concerning the side effects, one of the major goals of radiation research recently has been the development of a predictive in vitro assay. This paper is a contribution to that effort. MATERIALS AND METHODS: Skin fibroblasts of patients with normal-tissue reactions or acute and/or late increased side effects and cell lines of four individuals carrying the heritable disease ataxia telangiectasia were irradiated in vitro. The formation of micronuclei was chosen as biological endpoint and the results were compared with the clinically observed side effects. RESULTS: In general, a radiation dose-dependent increase in micronucleus frequency was found. The cells of the majority of the sensitive patients, as well as those of homozygous and heterozygous ataxia telangiectasia individuals, showed a higher micronucleus induction than the average of the donors with normal sensitivity. CONCLUSIONS: The micronucleus test seems to be a very promising tool in the evaluation of radiation sensitivity prior to therapy. However, larger studies are needed to confirm these findings and to optimize the methodology, and it is presumed that a final predictive assay will consist of a combination with other test systems.

Invasive thymoma: treatment with postoperative radiation therapy.

PURPOSE: To assess radiation therapy and chemotherapy in the treatment of invasive thymoma and thymic carcinoma. MATERIALS AND METHODS: In 1981-1995, 43 patients received irradiation after total (n = 23) or subtotal (n = 20) resection. Tumors were thymic carcinoma (n = 10) or invasive thymoma (n = 33). Masaoka stage was II in 10 patients, III in 14, and IV in 19. Median total dose applied was 50 Gy (range, 10-72 Gy). Seventeen patients (five with stage III and 12 with stage IV) also received chemotherapy. RESULTS: Patients with thymic carcinoma had a median survival of 9.5 months, compared with 50 months for patients with invasive thymoma (P = .008). Patients' median survival and 5-year survival rates were 97 months and 90% for stage II, 65 months and 67% for stage III, and 32.5 months and 30% for stage IV tumors (P = .024). Overall control rate within the radiation field was 81% (35 patients) and overall local control rate within the thorax was 74% (32 patients). Of the 17 patients who received chemotherapy and radiation therapy, nine had thymic carcinoma and a median survival of 12 months (range, 1.4-23.0 months). CONCLUSION: With total doses of 45-50 Gy, local control is achievable after radical resection. Whether patients with completely resected stage II thymomas should receive radiation after surgery remains uncertain, as does the role of chemotherapy in the treatment of thymoma.

[The locoregional recurrence of rectal carcinoma. A computed tomographic analysis and a target volume concept for adjuvant radiotherapy]. / Das lokoregionäre Rezidiv des Rektumkarzinoms. Eine computertomographische Analyse und ein Zielvolumenkonzept für die adjuvante Radiotherapie.

BACKGROUND: In the adjuvant postoperative radiotherapy of rectal carcinoma the knowledge of the predominant areas of recurrences is of major importance for the definition of the target volume. We analysed the pattern and locations of tumor recurrences in the CT scans of 155 patients and correlated the findings with the primary tumor location (above and below the peritoneal duplication) and the operating method (abdominoperineal extirpation, anterior resection. Hartmann procedure). PATIENTS AND METHOD: Hundred and fifty-five patients with the diagnosis of rectal carcinoma recurrences were treated in our institution between 1980 and 1995. To determine the extension of the recurrent tumor within the pelvic levels (presacral levels S1-S5, precoccygeal, pelvic floor level and perineal level) and the tumor infiltration of pelvic organs and muscles we analysed the pretherapeutic CT images. The lymph node recurrences were classified as: pararectal, presacral, iliac internal, iliac external, iliac communis and para-aortal recurrences. RESULTS: Sixty-one percent of the patients with rectum extirpation and 66% with anterior resection showed a combined local and nodal recurrence. Isolated lymph node recurrences were rare (4% and 5%) (Tables 2 and 3). The local recurrence was mostly situated in the presacral pelvis, predominantly there was an infiltration of the presacral space at the level of S4, S5 and os coccygis regardless of the operating method and the primary tumor location (Figure 1). The anastomosis was involved in the tumor recurrence in 93% of the anteriorly resected patients (Table 3). In 9 out of 96 patients after rectum extirpation the pelvic region caudal of the tip of the coccyx was the origin of the recurrent tumor (Table 2, Figure 2). Primarily all 9 patients had a deep-seated carcinoma (< 6 cm ab ano). Only 2 patients showed an isolated perineal recurrence after rectum extirpation (Table 2. Figure 2). Two thirds of the deep-seated tumors showed a vaginal involvement (Figures 3 and 4). The incidence of iliac internal and presacral nodal recurrence was 47 to 59% (Figures 3 and 4). The incidence of iliac external lymph node recurrences was 7% after rectum extirpation and 2% after anterior resection/Hartmann procedure. CONCLUSION: Our data demonstrate that 2/3 of the patients with tumor-bed recurrences also show lymph node recurrences predominantly in the iliac internal and presacral groups. This has to be considered in the definition of the boost target volume. The target volume must also include the dorsal wall of the urogenital organs. A ventral extension of target volume up to iliac external lymph nodes is not necessary.

Distinct vascular lesions in giant cell arteritis share identical T cell clonotypes.

Giant cell arteritis (GCA) is a spontaneous vasculitic syndrome that specifically targets the walls of medium and large arteries. Vascular lesions are characterized by patchy granulomatous infiltrates composed of T cells, macrophages, histiocytes, and giant cells. To test the hypothesis that a locally residing antigen recruits T cells into the vessel walls, we have analyzed T cell receptor (TCR) molecules of tissue infiltrating T cells. A total of 638 CD4+ T cell clones were isolated from temporal artery specimens of three patients with GCA. Analysis of TCR molecules for the usage of V beta 1-V beta 20 revealed that all TCR V beta elements were represented, demonstrating that interleukin 2 (IL-2)-responsive T cells infiltrating the tissue are highly diverse. To detect expanded T cell specificities, we made use of the patchy character of the inflammatory disease and compared the TCR repertoire of T cells established from independent vasculitic foci of the same artery. Sequence analysis of TCR V beta chains documented that individual TCR specificities were present in multiple copies, indicating clonal expansion. T cells with identical beta chains were isolated from distinct inflammatory foci of the same patient. These specificities represented only a small fraction of tissue-infiltrating T cells and involved the V beta 5.3 gene segment in the two patients sharing the HLA-DRB1*0401 allele. The third complementarity determining region of clonally expanded TCR beta chains was characterized by a cluster of negatively and positively charged residues, suggesting that the juxtaposed antigenic peptide is charged. The sharing of identical T cell specificities by distinct and independent regions of the granulomatous inflammation suggests that these T cells are disease relevant and that their repertoire is strongly restricted. These data suggest that an antigen residing in the arterial wall is recognized by a small fraction of CD4+ T cells in the inflammatory process characteristic for GCA.

Selection of T cell receptor V beta elements by HLA-DR determinants predisposing to rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is genetically linked to a sequence motif encoding for the middle portion of the alpha-helical loop, which is adjacent to the antigen-binding groove of the HLA-DR molecule. The disease-associated element might be involved in binding the antigen or in interacting with the T cell receptor (TCR). To investigate the contribution of the disease-associated element in T cell recognition events, we studied structural requirements in the interaction of T cell clones with HLA-DR determinants. METHODS: T cell clones restricted to disease-associated HLA-DR determinants were established by allogeneic stimulation of HLA-DRB1*0401+ or *0401- responders with HLA-DRB1*0404/8+ stimulators. Allele specific primer sets were used to identify the V beta gene segment expressed by individual clones. Sequence analysis was applied to study the diversity of the TCR beta-chain junctional regions. RESULTS: The repertoire of TCR V beta elements was strongly biased toward the usage of V beta 6. HLA-DRB1*0401+ and *0401- donors preferentially recruited V beta 6+ T cells to recognize the disease-associated HLA-DR determinant. Sequence data revealed that the V beta 6.6/7 and V beta 6.8/9 subtypes of the V beta 6 multigene family were overrepresented. The TCR beta chains were characterized by a high degree of junctional diversity, supporting the view that a multitude of peptide-DR complexes were recognized and that the preferential use of V beta 6 was dictated by the TCR beta chain-DR beta 1 chain contact. CONCLUSION: T cells reactive with the disease-associated HLA-DR structure are nonrandomly selected. The HLA-DR component predisposing to RA might define molecular requirements that restrict the TCR-HLA interaction. Thus, the phenomenon of HLA association in RA might reflect a genetic control of T cell recognition, through the selection of the TCR repertoire.

Clonal heterogeneity of superantigen reactivity in human V beta 6+ T cell clones. Limited contributions of V beta sequence polymorphisms.

Superantigens encoded in the genome or released by bacteria have been identified as potent modulators of the murine immune system. High frequencies of mature or immature T cells are activated or intrathymically deleted when superantigens cross-link MHC class II molecules and the V beta element of the TCR. The V beta specificity discriminates superantigens from polyclonal T cell stimulators as well as specific Ag and determines the immunomodulatory role in shaping the T cell repertoire. A similar regulatory function of superantigens in the human immune system is less well established. Here, we have studied a series of human T cell clones sharing the TCR V beta 6 element and describe a surprising heterogeneity in their responsiveness to staphylococcal exotoxins. The V beta 6 gene segment had the ability to respond to all staphylococcal enterotoxins (SE); however, for individual T cell clones, there was a clear predominance of SE C3 reactivity compared to SE B and SE C2. The clonal heterogeneity of SE responsiveness did not correlate to sequence polymorphisms in the fourth hypervariable region of the V beta 6 segment, the presumptive binding site for superantigens. Superantigen reactivity was crucially influenced by the presenting HLA-DR molecule, especially when the superantigen served as a coligand, enhancing or suppressing the Ag-specific activation of the TCR. These data suggest that the correlation between human TCR V beta gene segments and superantigen responses is not stringent. Potential intrathymic deletion mechanisms controlled by superantigens may be less selective in humans and may result in a leakiness influenced by the host HLA-DR molecules.